Reducing Risk of Contracting Clostridium-Difficile Associated Disease

ABSTRACT

A method of treating a patient to reduce risk of developing  Clostridium difficile -associated disease or reducing existing  Clostridium difficile -associated disease in a mammalian subject involves administering to a mammalian subject an effective amount of a germination-inhibiting compound derived from taurocholate. Novel compounds of this class are also provided.

RELATED APPLICATION DATA

This application claims priority from U.S. provisional PatentApplication Ser. No. 61/682,505, filed 13 Aug. 2012,having the sametitle, inventors and assignee as the present application.

GOVERNMENT RIGHTS

This invention was made with government support under CHE0957400 awardedby the National Science Foundation. The government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compounds which are of use in thetreatment of bacterial diseases and infections, to compositionscontaining those compounds and to methods of treating bacterial diseasesand infections using the compounds. In particular, the compounds of thepresent invention are useful for the treatment of infection with, anddiseases caused by, Clostridium difficile.

BACKGROUND TO THE INVENTION

Spore Germination Inhibiting Drugs and Clostridium difficile

The development of antibacterial drugs represents one of the mostimportant media advances of the 20^(th) Century. Previously untreatablediseases could now be readily controlled and it was felt that manydiseases would be eradicated with these new wonder drugs. Despite thesesignificant advances in treatment, infectious diseases are the thirdmajor use of mortality in the USA (Clin. Infect. Dis., 2004 38,12791286) and remain one of the most significant global healthcareproblems. Rates of resistance in all of the major pathogenic bacteriaare rising dramatically and of particular concern is the increasingnumber and severity a nosocomial infections Infectious Disease Societyof America, 2004, Bad Bugs, No Drugs). The emergence of multi-drugresistant pathogens has rendered many of the current frontline drugscompletely ineffective in controlling many diseases.

A particular subset of bacterial pathogens of concern is thoseclassified as spore-forming bacteria. Bacterial spores (endospores) aredormant, non-reproductive structures formed by bacteria in response toenvironmental stress. Once environmental conditions become favorable,the spores germinate and the bacteria proliferate. In the case ofpathogenic bacteria, germination in a human host may result in disease.Germination of a spore, such as Clostridium difficile is the process inwhich a spore begins to grow into vegetative cells, and sporelinghyphae.

Bacterial spores are extremely tolerant to many agents and environmentalconditions including radiation, desiccation, temperature, starvation andchemical agents. This natural tolerance to chemical agents allows sporesto persistent for many months in key environments such as hospitals,other healthcare centers and food production facilities, where standardcleaning agents, germicides and sterilization processes do not eradicatethe bacteria. In the case of food production, the presence of spores canhave significant consequences ranging from simple food spoilage to thespread of food-borne pathogens and food poisoning. More recently,attention has been drawn to the risks associated with the spores ofBacillus anthracis, the causative agent of anthrax. The spores can bereadily prepared as a dry powder that can be disseminated by numerousmethods and used as a bioterrorist agent. Anthrax is considered thesingle most worrying bioterrorism agent (CDC Emerg. Infect. Dis. 2004, 5(4), 552-555). This can be highlighted by the postal anthrax attacks inthe United States in 2001. There were 22 confirmed infections resultingin 5 deaths with the cost of cleanup and decontamination following theattacks estimated at $1 billion.

Important spore-forming bacteria are the Gram-positive endospore-formingbacteria of the genera Bacillus and Clostridium. Examples of the genusBacillus of health concern to humans include, but are not limited to, Banthracis and B. cereus. Bacillus anthracis is of particular concern asthe causative agent of anthrax. Anthrax infection can occur throughingestion, inhalation or cutaneous contact with Bacillus anthracisspores resulting in three distinct clinical forms. Cutaneous infectionaccounts for about 95% of all infections and is generally wellcontrolled with the use of suitable antibiotics. Around 20% of untreatedcases of cutaneous anthrax will result in death. Intestinal infection ischaracterized by an acute inflammation of the intestinal tract resultingin nausea, loss of appetite, vomiting, fever, abdominal pain, vomitingof blood and severe diarrhea. Intestinal anthrax results in death. in25% to 60% of cases. The most severe form of the disease is pulmonaryanthrax which is often fatal, even with aggressive and timely antibioticadministration. The ability to readily disperse anthrax spores throughthe air and over as wide area to induce pulmonary anthrax makes anthraxthe primary bioterrorism agent.

Members of the genus Clostridium are Gram-positive, spore-forming,obligate anaerobes. Exemplary species causing human disease include, butare not limited to, C. perfringens, C. tetani, C. botulinium, C.sordellii and C. difficile. Clostridia are associated with diverse humandiseases including tetanus, gas gangrene, botulism and pseudomembraneouscolitis and can be a causative agent in food poisoning.

Of particular concern is disease caused by Clostridium difficile.Clostridium difficile causes Clostridium difficile-associated diseases(CDAD) and there has been a ten-fold increase in the number of caseswithin the last 10 years, with hyper-virulent and drug resistant strainsnow becoming endemic. Recent Health Protection Agency (HPA) figures showthere were 55,681 cases of C. difficile infection in patients aged 65years and above in England in 2006 (up 8% from the previous year).Perhaps most worrying are the cases of CDAD now being reported with nounderlying antibiotic use

Clostridium difficile is a commensal enteric bacterium, the levels ofwhich are kept in check by the normal gut flora. However, the bacteriumis the causative agent of C. difficile associated disease (CDAD) and hasbeen identified as the primary cause of the most serious manifestationof CDAD, pseudomembraneous colitis. CDAD is associated with a wide rangeof symptoms ranging from mild diarrhea to pseudomembraneous colitis,toxic megacolon and death. The primary risk factor for the developmentof CDAD is the use of antibiotics disrupting the normal entericbacterial flora causing an overgrowth of Clostridium difficile. Althoughclindamycin is the major antibiotic associated with CDAD, the disease isnow associated with nearly all antibiotics including members of thefluoroquinolone, cephalosporin, macrolide, β-lactam and many othersclasses.

CDAD is primarily a concern in the hospital setting and is of particularconcern amongst elderly patients where mortality rates are particularlyhigh. Mortality rates in the USA have risen from 5.7 per million ofpopulation in 1999 to 23.7 per million in 2004. Colonization rates of C.difficile in the general population are up to 3% althoughhospitalization dramatically increases the rates of colonization up to25%. Of particular colleen) is the emergence anew endemic strains. Aparticularly pertinent example is the hyper-virulent BI/NAP1 (also knownas ribotype 027) strain which shows increased toxin A and B productionas well as the production of additional novel binary toxins. Thehyper-sporulation characteristics of strains such as BI/NAP1 contributesignificantly to the issue. Gastric acidity is part of the naturaldefense mechanism against ingested pathogens and any reduction in theacidity of the stomach can result in colonization of the normallysterile upper gastrointestinal tract which can result in a disturbanceof the normal enteric microflora. As such, the use of gastric acidsuppressive agents, such as proton pump inhibitors (PPIs) and histamineH2-receptor antagonists (H2RAs) is associated with an increased risk ofC. difficile colonization and subsequent development of CDAD. The use ofPPIs and H2RAs has previously been associated with other entericinfections such as traveller's diarrhea, salmonellosis and cholera. Ithas been reported that the risk of CDAD increases with the use ofgastric acid suppressive agents in both the community and hospitalsettings.

PPIs include, but are not limited to, omeprazole (Losec, Prilosec,Zegerid), lansoprazole (Prevacid, Zoton, Inhibitol), esomeprazole(Nexium), pantoprazole (Protonix, Somac, Pantoloc, Pantozol, Zurcal,Pan) and rabeprazole (Rabecid, Aciphex, Pariet, Rabeloc).

H2RAs include, but are not limited to, cimetidine (Tagamet), ranitidine(Zinetac, Zantac), famotidine, (Pepcidine, Pepcid), roxatidine (Roxit)and nizatidine (Tazac, Axid).

Triple therapy with PPIs or H2RAs together with a combination of twoantibiotics is a recognized treatment for the eradication ofHelicobacter pylori infections (Aliment. Pharmacol. Ther., 2001, 15(5),613-624; Helicobacter., 2005, 10(3), 157-171). However, there are a fewreports that this triple therapy regimen can lead to CDAD side effects(Am. J. Gastroenterel, 1998, 93(7), 1175-1176; J. Int. Med., 1998,243(3), 251-253; Aliment. Pharm. Ther., 2001, 15(9), 1445-1452; Med.Sci. Monit., 2001, 7(4), 751-754). Typical antibacterials used to treatHelicobacter pylori infections are a combination of agents selectedfrom, but not limited to metronidazole, amoxicillin, levofloxacin andclarithromycin—many of which are strongly associated with thedevelopment of CDAD. Current therapies are extremely limited;particularly in view of the fact nearly all antibiotic classes areassociated with causing the disease. The only FDA approved drug fortreatment of CDAD is vancomycin although metronidazole is alsoextensively used. Widespread vancomycin use for the treatment of CDAD isof concern due to its bacteriostatic action against clostridia,relatively high cost and the possible selection of resistant C.difficile strains as well as other bacteria (particularly Enterococcusspp.). A key issue with both metronidazole and vancomycin is the highrelapse rate with at least 20% of patients experiencing at least onerecurrent episode. Relapse is proposed to occur due to the inability toeradicate the clostridium spores during therapy resulting in subsequentoutgrowth to a pathogenic state. This inability to control sporeformation allows for continued contamination of the hospitalenvironment. As such, agents able to eradicate vegetative cells andcontrol endospores would be of significant advantage.

The primary therapy option for the treatment of CDAD is discontinuationof any current antimicrobial treatment followed by appropriate use ofeither vancomycin or metronidazole. Both agents are usually administeredorally although metronidazole may also be administered intravenously andin severe cases, vancomycin may also be administered via numerous othermutes including intracolonic, through nasal gastric tube or as avancomycin-retention enema. Additional antibiotics agents that have beenreported to be used in the treatment of CDAD include fusidic acid,rifamycin and its analogues, teicoplanin and bacitracin although noneshow particular efficacy over vancomycin or metronidazole. In additionto halting any offending antibacterial treatment, the use ofantiperistaltic agents, opiates, or loperamide should be avoided sincethey can reduce clearance of the C. difficile toxins and exacerbatetoxin-mediated colonic injury.

Alternative therapies, used as stand-alone agents or in conjunction withantibacterials, are aimed at either trying to re-establish the nativegut microorganism population, reducing the levels of C. difficile toxinsor stimulating the immune system. Thus, alternative CDAD therapiesinclude provision of Saccharomyces boulardii or Lactobacillusacidophilus in conjunction with antibiotics, faecal transplantation andin severe cases where all other therapy options have failed, surgery.Although rates of colectomy are low (up to 3% of cases) it is associatedwith high mortality rates (up to 60%).

As such, there is a pressing need for new and effective agents to treatdiseases associated with spore forming bacteria, particularly thosecaused by members of the genera Clostridium and Bacillus and inparticular disease associated with Clostridium difficile infection. Thisneed is particularly acute in the light of the refractory nature ofClostridium difficile to many broad spectrum antibiotics (includingβ-lactam and quinolone antibiotics) and the frequency with whichresistance emerges.

Among the Prior Art relevant to addressing issues with Clostridiumdifficile are:

WO2007056330, WO2003105846 and WO2002060879 disclose various 2-aminobenzimidazoles as antibacterial agents.

WO2007148093 discloses various 2-amino benzothiazoles as antibacterialagents.

WO2006076009, WO2004041209 and Bowser et at (Bioorg. Med. Chem. Lett.,2007, 17, 5652-5655) disclose various substituted berry compounds usefulas anti-infectives that decrease resistance, virulence, or growth ofmicrobes. The compounds are said not to exhibit intrinsic antimicrobialactivity in vitro.

U.S. Pat. No. 5,824,698 discloses various dibenzimidazoles asbroad-spectrum antibiotics, disclosing activity against bothGram-negative and Gram-positive bacteria, including Staphylococcus app.and Enterococcus app. However, this document does not disclose activityagainst anaerobic sport-forming bacteria and in particular does notdisclose activity against any Clostridium spp. (including C. difficile).

US Published Patent Application Document No. 200701 12048 (Bavari)discloses various bi- and triarylimidazolidines and bi- andtriarylamidines as broad-spectrum antibiotics, disclosing activityagainst both Gram-negative and Gram-positive bacteria, includingStaphylococcus spp., Enterococcus spp. and Clostridium spp. However,this document does not disclose compounds of general formula (I) asdescribed herein.

It has been found that certain imidazoles and or their derivatives arecapable of inhibiting the growth of Clostridium difficile (George,1979), MRSA (Lee & Kim, 1999) and/or VISA, VRSA and VRE. However, theidentification of compounds that act synergistically with these drugs(the imidazoles) means that lower concentrations of original drug may beused (thus reducing the undesirable side effects of the imidazoles) andprolonging the life of the drug treatment (e.g. a synergisticcombination of two drugs will require resistance to develop in bothcomponents before the combination becomes ineffective). If thespontaneous rate of resistance development in an organism is 10⁸, thedevelopment of resistance to the combination of two compounds will beapproximately 10⁶⁴, therefore the risk of resistance developing isdramatically lower.

US Published Patent Application Document No. 20110229583 (Tran)describes that a medicinal drug is administered to a person for treatinga medical condition of the person or/and for preventing the person fromcontracting the medical condition. The medical condition can be abacterial infection, a eukaryotic infection, a prion-caused infection, anonpathogenic inflammation, and, insofar as not covered by any of thesefour types of the medical condition, a fungal infection, a spore-causedinfection, and a parasitic infection. A medicinal drug is similarlyadministered non-topically to a person for treating a virus-causedmedical condition of the person or/and for preventing the person fromcontracting the virus-caused medical condition. The medicinal drug istypically formed at least partially with salt of peroxymonosulfuricacid, preferably potassium hydrogen peroxyanonosulfate.

US Published Patent Application Document No. 20110183360 (Rajagapol)describes an isolated antibody that binds to putativeN-acetylmuramoyl-L-alanine amidase protein of Clostridium difficilestrain 630 having SEQ ID NO: 5 or a fragment of the putativeN-acetylmuramoyl-L-alanine amidase protein. In some embodiments, theisolated antibody binds to a fragment of the putativeN-acetylmuramoyl-L-alanine amidase protein including amino acid residues294 to 393. In some embodiments, the isolated antibody binds to afragment of the putative N-acetylmuramoyl-L-alanine amidase proteinincluding amino acid residues 582 to 596. In some embodiments, theisolated antibody binds to a fragment of the putativeN-acetylmuramoyl-L-alanine amidase protein including amino acid residues64 to 78.

Published U.S. Patent Application Document No. 20110086797 (Dworkin)describes compositions and methods for treating bacterial infections. Itis demonstrated herein that bacteria cell wall materials stimulategermination of spores of Gram-positive bacteria, and that such activityrequires Ser/Thr kinase PrkC. By modulating one or both, spores (whichcan be antibiotic resistant) can be stimulated or inhibited fromgermination, which can be exploited in various methods of therapeutictreatment. Also provided is a method of modulating germination of aspore of a Gram-positive bacterium. Also provided is a method ofdecontaminating an environment.

Published U.S. Patent Application Document No. 20120020950 (Davis)describes novel compounds of a specific formula (I), which are of use inthe treatment of bacterial diseases and infections, to compositionscontaining those compounds and to methods of treating bacterial diseasesand infections using the compounds. In particular, the compounds areuseful for the treatment of infection with, and diseases caused by,Clostridium difficile.

Published U.S. Patent Application Document No. 20080254010 (Sasser)discloses treating a patient infected with spore-forming bacteria byadministering to the patient an antibiotic and a spore germinant inamounts and for durations effective for treating said patient. Among thespore germinants is listed bile salts, and specifically taurocholate.

Published U.S. Patent Application Document No. 20110280847 (Sorg)describes methods and treatments for inhibiting Clostridium difficilespore germination and outgrowth using chemical means. Among the chemicalmeans are specific compounds derivatized from specific bile saltsdefined by structural formulae.

SUMMARY OF THE INVENTION

A method of reducing risk of developing Clostridium difficile-associateddisease or reducing existing Clostridium difficile-associated disease ina mammalian subject receiving antibiotic therapy, comprisingadministering to a mammalian subject an effective amount of a compoundderived from taurocholate. The compound derived from taurocholatepreferably has the central core structure of the formula:

The method may include parallel or adjacent or preceding or subsequenttreatment with an antibiotic, with the mammalian subject alreadyreceiving antibiotic therapy and at risk of developing C.difficile-associated disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Germination kinetic graphs showing agonistic and antagonisticbehavior of molecules with C. difficile spores.

FIG. 2. Amino acids assessed for activation or inhibition ofglycine-mediated germination in C. difficile spores.

FIG. 3. Comparison of amino acids as agonists of C. difficile sporegermination.

FIG. 4. Germination kinetic graph showing behavior of C. difficilespores and germinants in buffer and complex media.

FIG. 5. A chart of taurocholate analogs assessed for activation orinhibition of taurocholate mediated germination in C. difficile spores.

FIG. 6 is a generic structural formula showing the central core and ringpositions on a taurocholate molecule.

FIG. 7 is a more specific central core nucleus showing at least themajor positions on the taurocholate central core on with substituentsare placed within the scope of the present technology.

FIG. 8 is Table 3. Effect of taurocholate hydroxyl groups on C.difficile spore germination.

FIG. 9 is Table 4. Effect of the taurocholate side chain on C. difficilespore germination.

FIG. 10 is a graph evidencing that mice treated with CamSA orchenodeoxycholate show no weight changes with particular treatments.

FIG. 11 shows graphic results of protection of mice from CDI bydifferent bile salts. The data is shown in a Kaplan-Meier survival plotfor treated C. difficile infected mice.

FIG. 12 shown graphic representations of signs of severity for C.difficile infected animals treated with different bile salts.

FIG. 13 graphically shows that CamSA does not affect vegetativebacterial growth.

FIG. 14 graphically shows that CamSA is not toxic to mammalian cells.

FIG. 15 graphically shows distribution of C. difficile spores in the GItract of CamSA-treated animals. The stomach (St), duodenum (Du), jejunum(Je), and ileum (II) showed negligible amounts of spores compared to thecert (Cc) and colon (Co).

FIG. 16 is graphic evidence that CamSA protects mice from CDI.

FIG. 17 is a graphic representation of data showing that C. difficilespores accumulate in the cecum, colon, and feces of CamSA-treatedanimals.

FIG. 18 is a time line model for CDI onset in mice. C. difficile spores(black circles) that are ingested by the host.

FIG. 19 is a graphic representation of the stability of CamSA andtaurocholate towards bile salt hydrolases.

FIG. 20 is a graphic representation of the cytotoxicity of CamSA.

FIG. 21 is a graphic representation of inhibition of C. difficile toxinproduction by CamSA treatment.

FIG. 22 is a graphic representation that CDI is established between 6and 9 hours post-infection.

DETAILED DESCRIPTION OF THE INVENTION

Novel compounds and a method of using those novel compounds and evenknown compounds in a treatment for mammalian subjects. The treatment mayaffect (reduce) the rate of germination of spores that form Clostridiumdifficile and may reduce risk of developing Clostridiumdifficile-associated disease or reducing existing Clostridiumdifficile-associated disease in a mammalian subject receiving antibiotictherapy, comprising administering to a mammalian subject an effectiveamount of a compound derived from taurocholate.

The term “central core structure” is intended to mean thatpharmacologically acceptable dertivatives of the core structure shown inthe formula are included within the scope of that description, whetherin the specific or the claims. Only there reference is specific to aformula, without expensive legal or technical terms is a narrowerconstruction of the scope of a formula intended.

The methods of the present technology and the included compounds may beapproximately generally include a method of vesting a patient to reducerisk of developing Clostridium difficile-associated disease or reducingexisting Clostridium difficile-associated disease in a mammaliansubject. Steps may include administering to a mammalian subject aneffective amount of a germination-inhibiting compound derived fromtaurocholate. The compound derived from taurocholate may, for example,have a central core structure of the formula:

The compound derived from taurocholate may be, for example, a compoundwithin the non-limiting, exemplary formula:

wherein R¹ is selected from the group consisting of hydrogen, loweralkyl of C₁ to C₆, amines, halogen, cyano, hydroxyl, carboxylic acidgroups and substituted lower alkyl of C₁ to C₆, wherein the substituentsare selected from the group consisting of halogen, cyano, hydroxyl andthe like;R² is selected from the group consisting of hydrogen, lower alkyl of C₁to C₆, halogen, cyano, amines, hydroxyl, carboxylic acid groups andsubstituted lower alkyl of C₁ to C₆, wherein the substituents areselected from the group consisting of halogen, cystic, hydroxyl and thelike. R³ is selected from the group consisting of hydrogen, lower alkylof C₁ to C₆, halogen, cyano, hydrogen, carboxylic acid groups andsubstituted lower alkyl of C₁ to C₆, wherein the substituents on thealkyl groups are selected from the group consisting of halogen, cyano,hydroxyl;R⁴ is selected from the group consisting of O and S; andR⁵ is selected from the group consisting of

1) NH(CH₂)₂SO₃H

2) NHCH₂SO₃H

3) NH(CH₂)₃SO₃H

4) NH(p-(C₆H₄))SO₃H

5) NH(o-(C₆H₄))SO₃H

6) NH(m-(C₆H₄)SO₃H

7) NH(CH₂)₂SO₂H

8) NHCH₂CO₂H

9) NH(CH₂)₂CO₂H

10) NH(CH₂)₂CONH(CH₂)₂CO₂H

11) NH(CH₂)₃CO₂H

12) NH(CH₂)₃CONH(CH₂)₃CO₂H

13) O(CH₂)₂SO₃H

Compound Modifications to EC₅₀ ^(a) IC₅₀ ^(b) No. taurocholate sidechain (stdev) mM (stdev) mM 14 NH(CH₂)₄COOH NA 5.3 (0.24) 15NH(CH₂)₄CONH(CH₂)₄COOH NA NA 16 NH(CH₂)₅COOH NA 2.3 (0.075) 17NH(CH₂)₅CONH(CH₂)₅COOH NA NA 18 NH(CH₂)₂OSO₃H NA NA 19 S(CH₂)₂SO₃H NA NA20 NHCH₂PO₃H NA NA 21 NHC₆H₅ NA 0.27 (0.070) 22 NHC₅H₄N NA NA 23NH(m-C₆H₄))COOH NA 3.1 (0.78) 24 NH(o-(C₆H₄))COOH NA NA 25NH(p-(C₆H₄))COOH NA 1.4 (0.11) 26 NH(m-(C₅H₈))COOH NA 0.47 (0.069) 27NH(m-(C₆H₄))COOCH₃ NA NA 28 NH(m-(C₆H₄))OPO₃H₂ NA NA 29 NH(m-(C₆H₄))OHNA NA 30 NH(o-(C₆H₄))OH NA NA 31 NH(m-(C₆H₄))SH NA NA 32 NH(p-(C₆H₄))SHNA NA 33 NH(m-(C₆H₄))SCH₃ NA NA 34 NH(m-(C₆H₄))NH₂ NA 0.53 (0.022) 35NH(m-(C₆H₄))CH₃ NA NA 36 NH(o-CH₃-m-(C₆H₄)SO₃H NA NA 37NH(o-OCH₃-m-(C₆H₄)SO₃H 4.6 (0.34) NA 38 NH(o-CH₃-m-(C₆H₄)COOH 16 (0.90)NA 39 NH(p-CH₃-m-(C₆H₄)COOH NA 11 (0.025) 40 NH(o,p-(CH₃)₂-m-(C₆H₄)SO₃HNA 5.6 (0.042) 41 NH(p,m-(CH₃)₂-m-(C₆H₄)SO₃H NA 1.1 (0.098) 42NCH₃(m-(C₆H₄)SO₃H 5.4 (0.10) NA 43 NH₂(m-(C₆H₄)SO₃H NA NA 44NHCH₃(m-(C₆H₄)SO₃H NA NA 45 NH(1-C₁₀H₈) NA NA 46 NH(2-C₁₀H₈) NA NA 47NH(C₁₄H₁₀) NA NA 48 NH(C₁₆H₁₀) NA NA 49 NH(C₁₀H₈)-3-OH NA NA 50NH(C₁₀H₈)-1-SO₃H NA NA 52 NH(C₁₀H₈)-1-CO₂H NA NA 53 NH(C₁₀H₈)-5-CO₂H 1.3(0.19) NA 54 NH(C₁₀H₈)-3-OH-5-SO₃H NA NA 55 NH(C₁₀H₈)-1,6-SO₃H NA NA 56NH(C₁₀H₈)-2,6-SO₃H 5.7 (0.26) NA 57 NH₂(C₁₀H₈)-1,5-SO₃H NA NA CompoundModifications to EC50 IC50 No. taurochenodeoxycholate side chain (stdev)mM (stdev) mM 58 NHCH₂SO₃H 0.31 (0.023) NA 59 NH(CH₂)₂SO₂H NA 5.3 (0.20)60 NH(CH₂)₂COOH NA 3.9 (0.80) 61 NH(CH₂)₄COOH NA 0.64 (0.19) 62NH(CH₂)₄CONH(CH₂)₄COOH NA 1.9 (0.78) 63 NH(CH₂)₅COOH NA 1.1 (0.23) 64NH(CH₂)₅CONH(CH₂)₅COOH NA 8.6 (0.38) 65 NH(m-(C₆H₄))SO₃H NA 6.5 (0.20)66 NHC₆H₅ NA NA 67 NH(m-(C₆H₄))COOH NA 0.93 (0.12) 68 NH(p-(C₆H₄))COOHNA NA 69 NH(m-(C₆H₈))COOH 7.4 (0.65) NA 70 NH(m-(C₆H₄))NH₂ NA NA 71NH(p-CH₃-m-(C₅H₄)COOH NA 0.092 (0.014) 72 NH(o,p-(CH₃)₂-m-(C₆H₄)SO₃H NA0.92 (0.16) 73 NH(p,m-(CH₃)₂-m-(C₆H₄)SO₃H NA 0.75 (0.077) 74NH(C₁₀H₈)-2,6-SO₃H NA 12 (0.48) Compound Modifications to EC50 IC50 No.cholic acid (stdev) mM (stdev) mM 75 Shorter alkyl chain NA 6.3 (0.0070)76 Alcohol side chain NA 0.33 (0.0072) 77 Methyl ester side chain NA0.059 (0.0079) 78 Methyl ester side chain - shorter alkyl NA 0.15(0.063) chain by 1 carbon 79 Methyl ester side chain - shorter alkyl NA0.66 (0.10) chain by 2 carbon 80 Ethyl ester side chain NA 0.0082(0.00050) 81 Methoxylated hydroxyl groups NA NA 82 Hydroxyls at 3 (α)and 12 (α) NA 0.19 (0.025) 83 Hydroxyls at 3 (α) and 12 (β) NA NA 84Hydroxyls at 3 (β) and 12 (α) NA 0.78 (0.073) 85 Methyl ester withhydroxyls at 3 (α) and NA 0.097 (0.068) 12(α) 86 Methyl ester withhydroxyls at 3 (α) NA 0.095 (0.0012) and 12 (β) 87 Hydroxyls at 3 (α)and 6 (α) NA 1.24 (0.11) 88 Methyl ester with hydroxyls at 3 (α) and NA0.037 (0.021) 6 (α) 89 Alcohol with hydroxyl at 3 (α) NA NA 90 Methylester with hydroxyl at 12 (α) NA 0.37 (0.051) 91 Methyl ester withhydroxyl at 3 (β) NA 1.3 (0.54) ^(a) C. difficile spores wereindividually treated with 6 mM glycine and bile salt analogs. Standarddeviations are shown in parentheses. ^(b) C. difficile spores wereincubated with bile salt analogs for 15 min prior to the addition of 6mM taurocholate and 12 mM glycine. Standard deviations are shown inparentheses. ^(c) NA, no change in absorbance after 90 minutes under theconditions tested thus no statistics could be performed.

The present technology evidences that inhibiting Clostridium difficilespore germination serves as a prophylactic approach to preventClostridium difficile-associated diseases (CDAD) or Clostridiumdifficile infection (CDI). CDI is the major cause ofantibiotic-associated diarrhea. In the US, there are approximately500,000 CDI cases annually, with a mortality rate >2.5%. The annualCDI-associated costs have been estimated at $3.2 billion. CDI respondspoorly to most antibiotics since its onset typically occurs whilepatients are receiving antimicrobial therapy. Indeed, only metronidazoleand vancomycin are currently used to treat CDI. The incidence of CDI iscomplicated by the appearance of highly resistant and hypervirulentstrains.

The infectious form of C. difficile is the spore, a dormant andresistant structure formed from vegetative cells during nutrientdeprivation. C. difficile spores revert to toxin-producing bacteria (aprocess called germination) in nutrient-rich environments. Germinationof C. difficile spores in the GI tract of hospitalized patients is thefirst committed step in CDI. We propose that compounds able to curtailspore germination could also prevent CDI establishment. Anti-germinationgermination compounds could be used to supplement antibiotic treatmentsof hospitalized patients. Once the antibiotic regime is completed,re-establishment of the normal gut flora will prevent C. difficilecolonization and anti-germination therapy can also be stopped. Thepresent technology evidences that C. difficile spores bind thegerminants taurocholate (a bile salt) and glycine through a complexmechanism. We also found that a synthetic taurocholate analog with anm-aminobenzenesulfonic acid side chain (CamSA) is an efficient inhibitorof C. difficile spore germination. CamSA binds to C. difficile sporesapproximately 1,000-fold better than the natural taurocholate germinant.More recently, we found that CamSA (an anti-germinant) prevents CDAD ina mouse model, while taurocholate itself (a germinant) worsen CDIsymptoms. Even more, CamSA does not show acute toxicity even at 300mg/Kg and is stable to the GI tract environment.

The transformation process front a dormant spore to a fully vegetativebacterium is the initial step in CDI infections. New methods to impedespore germination would preclude the production of toxins andantibiotic-resistant factors, thus reducing morbidity and mortality.Targeting spore germination to prevent CDI is an approach that couldcomplement future antibiotic treatments. Since the first step in theestablishment of CDI is the germination of C. difficile spores in themicroflora-depleted gut of hospitalized patients, anti-germinationcompounds (e.g. CamSA and CamSA analogs) could be used in combinationtherapies to supplement antibiotic treatments in immune-compromisedpatients. Once the antibiotic regime is completed, re-establishment ofthe normal gut flora will prevent C. difficile spore germination andanti-germination therapy can also be stopped.

The method may include parallel or adjacent or preceding or subsequenttreatment with an antibiotic, with the mammalian subject alreadyreceiving antibiotic therapy and at risk of developing C.difficile-associated disease.

As used herein, the term “disease” is used to define any abnormalcondition that impairs physiological function and is associated withspecific symptoms. The term is used broadly to encompass any disorder,illness, abnormality, pathology, sickness, condition or syndrome inwhich physiological function is impaired irrespective of the nature ofthe aetiology (or indeed whether the aetiological basis for the diseaseis established). It therefore encompasses conditions arising fromtrauma, injury, surgery, radiological ablation, poisoning or nutritionaldeficiencies.

As used herein, the term “bacterial disease” refers to any disease thatinvolves (e.g. is caused, exacerbated, associated with or characterizedby the presence of) a bacterium residing and/or replicating in the bodyand/or cells of a subject. The term therefore includes diseases causedor exacerbated by bacterial toxins (which may also be referred to hereinas “bacterial intoxication”).

As used herein, the term Clostridium difficile-associated disease (CDAD)or Clostridium difficile infection (CDI) is used to define any diseasethat involves (e.g. is caused, exacerbated, associated with orcharacterized by the presence of) Clostridium difficile residing and/orreplicating in the body of a subject. Thus, the term covers any disease,disorder, pathology, symptom, clinical condition or syndrome in whichbacteria of the species Clostridium difficile act as aetiological agentsor in which infection with one or more strains of Clostridium difficileis implicated, detected or involved. The term therefore includes thevarious forms of colitis, pseudomembranous colitis, diarrhea andantibiotic-associated disease.

As used herein, the term “bacterial infection” is used to define acondition in which a subject is infected with a bacterium. The infectionmay be symptomatic or asymptomatic. In the latter case, the subject maybe identified as infected on the basis of various tests, including forexample biochemical tests, serological tests, microbiological cultureand/or microscopy.

The terms bacteriostatic and bacteriocidal are terms of art used todefine the ability to prevent (or reduce the rate of) bacterial growthand to mediate (directly or indirectly) the cellular destruction ofbacterial cells, respectively. The terms are not mutually exclusive, andmany agents exert both bacteriostatic and bacteriocidal effects (in somecases in a dose-specific or target-specific manner). In general,bacteriocidal agents yield better therapeutic results and are preferred.

As used herein, the term “broad spectrum antibiotic” defines an agentwhich is bacteriocidal and/or bacteriostatic for a range of bacteriaincluding both Gram-positive and Gram-negative bacteria.

The “term multi-drug resistant” (MDR) as applied herein to a bacteriumdefines a bacterium which is resistant to two or more classes ofantibiotics including, but not limited to, antibiotics selected frompenicillin, methicillin, quinolone, macrolide and/or vancomycin.

As used herein, the term “treatment” or “treating” refers to anintervention (e.g., the administration of an agent to a subject) whichcures, ameliorates, stabilizes or lessens the symptoms of a disease orremoves (or lessens the impact of) its cause(s) (for example, thecausative bacterium). In this case, the term is used synonymously withthe term “therapy.” Thus, the treatment of infection according to theinvention may be characterized by the (direct or indirect)bacteriostatic and/or bacteriocidal action of the compounds of theinvention.

Additionally, the terms “treatment” or “treating” refers to anintervention (e.g., the administration of an agent to a subject) whichprevents, slows or delays the onset or progression of a disease orreduces (or eradicates) its incidence within a treated population. Inthis case, the term treatment is used synonymously with the term“prophylaxis.”

The term subject (which is to be read to include “individual”, “animal”,“patient” or “mammal” where context permits) defines any subject,particularly a mammalian subject, for whom treatment is indicated.Mammalian subjects include, but are not limited to humans, domesticanimals, farm animals, zoo animals, sport animals, pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows;primates such as apes, monkeys, orangutans, and chimpanzees; canids suchas dogs and wolves; felids such as cats, lions, and tigers; equids suchas horses, donkeys, and zebras; food animals such as cows, pigs, andsheep; ungulates such as deer and giraffes; rodents such as mice, rats,hamsters and guinea pigs; and so on. In preferred embodiments, thesubject is a human, for example an infant human.

The term Gram-positive bacterium is a term of art defining a particularclass of bacteria that are grouped together on the basis of certain cellwall staining characteristics.

The term low G+C Gram-positive bacterium is a term of art defining aparticular subclass class of evolutionarily related bacteria within theGram-positives on the basis of the composition of the bases in the DNA.The subclass includes Streptococcus spp., Staphylococcus spp., Listeriaspp., Bacillus spp., Clostridium spp., Enterococcus spp. andLactobacillus spp.

The term “minimum inhibitory concentration” or “MIC” defines the lowestconcentration of a test compound that is needed to inhibit germinationof a spore in vitro or in vivo. A common method for determining the MICof an antibiotic is to prepare several tubes containing serial dilutionsof the test compound that are then inoculated with the bacterial isolateof interest. Following incubation at appropriate atmosphere andtemperature, the MIC of an antibiotic can be determined from the tubewith the lowest concentration that shows no turbidity.

As used herein, the term “combination”, as applied to two or morecompounds and/or agents (also referred to herein as the components), isintended to define material in which the two or more compounds/agentsare associated. The terms “combined” and “combining” in this context areto be interpreted accordingly. The association of the two or morecompounds/agents in a combination may be physical or non-physical.Examples of physically associated combined compounds/agents include:compositions (e.g. unitary formulations) comprising the two or morecompounds/agents in admixture (for example within the same unit dose);compositions comprising material in which the two or morecompounds/agents are chemically/physicochemically linked (for example bycrosslinking, molecular agglomeration or binding to a common vehiclemoiety); compositions comprising material in which the two or morecompounds/agents are chemically/physicochemically co-packaged (forexample, disposed on or within lipid vesicles, particles (e.g., micro-or nanoparticles) or emulsion droplets); pharmaceutical kits,pharmaceutical packs or patient packs in which the two or morecompounds/agents are co-packaged or co-presented (e.g. as part of anarray of unit doses).

Examples of non-physically associated combined compounds/agents include:

material (e.g. a non-unitary formulation) comprising at least one of thetwo or more compounds/agents together with instructions for theextemporaneous association of the at least one compound/agent to form aphysical association of the two or more compounds/agents;

material (e.g. a non-unitary formulation) comprising at least one of thetwo or more compounds/agents together with instructions for combinationtherapy with the two or more compound/agents;

material comprising at least one of the two or more compounds/agentstogether with instructions for administration to a patient population inwhich the other(s) of the two or more compounds/agents have been or arebeing) administered;

material comprising at least one of the two or more compounds/agents inan amount or in a form which is specifically adapted for use incombination with the other(s) of the two or more compounds/agents.

As used herein, the term “combination therapy” is intended to definetherapies which comprise the use of a combination of two or morecompounds/agents (as defined above) whether both are germinationsuppressants or one is an antimicrobial, such as an antibiotic orantifungal agent. Thus, references to “combination therapy,”“combinations” and the use of compounds/agents “in combination” in thisapplication may refer to compounds/agents that are administered as partof the same overall treatment regimen. As such the posology of each ofthe two or more compounds/agents may differ each may be administered atthe same time or at different times. It will therefore be appreciatedthat the compounds/agents of the combination may be administeredsequentially (e.g. before or after) or simultaneously, either in thesame pharmaceutical formulation (i.e. together), or in differentpharmaceutical formulations (i.e. separately). Simultaneously in thesame formulation is as a unitary formulation, whereas simultaneously indifferent pharmaceutical formulations is non-unitary. The posologies ofeach of the two or more compounds/agents in a combination therapy mayalso differ with respect to the route of administration.

As used herein, an effective amount or a therapeutically effectiveamount of a compound defines an amount that can be administered to asubject without excessive toxicity, irritation, allergic response, orother problem or complication, commensurate with a reasonablebenefit/risk ratio, but one that is sufficient to provide the desiredeffect, e.g., the treatment or prophylaxis manifested by a permanent ortemporary improvement in the subject's condition. The amount will varyfrom subject to subject, depending on the age and general condition ofthe individual, mode of administration and other factors. Thus, while itis not possible to specify an exact effective amount, those skilled inthe art will be able to determine an appropriate ‘effective’ amount inany individual case using routine experimentation and background generalknowledge. A therapeutic result in this context includes eradication orlessening of symptoms, reduced pain or discomfort, prolonged survival,improved mobility and other markers of clinical improvement Atherapeutic result need not be a complete cure.

As used herein, a “prophylactically effective amount” refers to anamount effective, at dosages, at particular sites and for periods oftime necessary, to achieve a prophylactic result or the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The term “efficacious” includes advantageous effects such as additivity,synergism, reduced side effects, reduced toxicity or improvedperformance or activity. Advantageously, an efficacious of may allow forlower doses of each or either component to be administered to a patient,thereby decreasing the toxicity, whilst producing and/or maintaining thesame therapeutic effect. A synergistic effect in the present contextrefers to a therapeutic effect produced by the combination which islarger than the sum of the therapeutic effects of the components of thecombination when presented individually. An additive e rot in thepresent context refers to a therapeutic effect produced by thecombination which is larger than the therapeutic effect of any of thecomponents of the combination when presented individually.

The term “ancillary compound” (or “ancillary agent”) as used herein isintended to define any compound which yields an efficacious combination(as herein defined) when combined with a compound of the invention. Theancillary compound may therefore act as an adjunct to the compound ofthe invention, or may otherwise contribute to the efficacy of thecombination (for example, by producing a synergistic or additive effector by potentiating the activity of the compound of the invention).

The term “adjunctive” as applied to the use of the compounds andcombinations of the invention in therapy or prophylaxis defines uses inwhich the materials are administered together with one or more otherdrugs, interventions, regimens or treatments (such as surgery and/orirradiation). Such adjunctive therapies may comprise the concurrent,separate or sequential administration/application of the materials ofthe invention and the other treatment(s). Thus, in some embodiments,adjunctive use of the materials of the invention is reflected in theformulation of the pharmaceutical compositions of the invention. Forexample, adjunctive use may be reflected in a specific unit dosage or informulations in which the compound of the invention is present inadmixture with the other drug(s) with which it is to be usedadjunctively (or else physically associated with the other drugs) withina single unit dose. In other embodiments, adjunctive use of thecompounds or compositions of the invention may be reflected in thecomposition of the pharmaceutical kits of the invention, wherein thecompound of the invention is co-packaged (e.g., as part of an array ofunit doses) with the other drug(s) with which it is to be usedadjunctively. In yet other embodiments, adjunctive use of the compoundsof the invention may be reflected in the content of the informationand/or instructions co-packaged with the compound relating toformulation and/or posology.

The term pharmaceutically acceptable derivative as applied to thecompounds of the invention define compounds which are obtained (orobtainable) by chemical derivatization of the parent compounds of theinvention. The pharmaceutically acceptable derivatives are thereforesuitable for administration to or use in contact with mammalian tissueswithout undue toxicity, irritation or allergic response (i.e.,commensurate with a reasonable benefit/risk ratio). Preferredderivatives are those obtained (or obtainable) by alkylation,esterification or acylation of the parent compounds of the invention.The derivatives may be active per se, or may be inactive until processedin vivo. In the latter case, the derivatives of the invention act asprodrugs. Particularly preferred prodrugs are ester derivatives whichare esterified at one or more of the free hydroxyls and which areactivated by hydrolysis in vivo. Other preferred prodrugs are covalentlybonded compounds which release the active parent drug according togeneral formula (I) after cleavage of the covalent bond(s) vivo.

The pharmaceutically acceptable derivatives of the invention retain someor all of the activity of the parent compound. In some cases, theactivity is increased by derivatization. Derivatization may also augmentother biological activities of the compound, for examplebioavailability.

The term pharmaceutically acceptable salt as applied to the compounds ofthe invention defines any non-toxic organic or inorganic acid additionsalt of the free base compound which is suitable for use in contact withmammalian tissues without undue toxicity, irritation, allergic responseand which are commensurate with a reasonable benefit/risk ratio.Suitable pharmaceutically acceptable salts are well known in the art.Examples are the salts with inorganic acids (for example hydrochloric,hydrobromic, sulfuric and phosphoric acids), organic carboxylic acids(for example acetic, propionic, glycolic, lactic, pyruvic, malonic,succinic, fumaric, malic, tartaric, citric, ascorbic, maleic,hydroxymaleic, dihydroxymaleic, benzoic, phenylacetic, 4-aminobenzoic,4-hydroxybenzoic, anthranilic, cinnamic, salicylic, 2-phenoxybenzoic,2acetoxybenzoic and mandelic acid) and organic sulfonic acids (forexample methanesulfonic acid and p-toluenesulfonic acid). The compoundsof the invention may also be converted into salts by reaction with analkali metal halide, for example sodium chloride, sodium iodide orlithium iodide. Preferably, the compounds of the invention are convertedinto their salts by reaction with a stoichiometric amount of sodiumchloride in the presence of a solvent such as acetone.

These salts and the free base compounds can exist in either a hydratedor a substantially anhydrous form. Crystalline forms of the compounds ofthe invention are also contemplated and in general the acid additionsalts of the compounds of the invention are crystalline materials whichare soluble in water and various hydrophilic organic solvents and whichin comparison to their free base forms, demonstrate higher inchingpoints and an increased solubility.

The term pharmaceutically acceptable solvate as applied to the compoundsof the invention defines any pharmaceutically acceptable solvate form ofa specified compound that retains the biological effectiveness of suchcompound. Examples of solvates include compounds of the invention incombination with water (hydrates), isopropanol, ethanol, methanol,dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine, oracetone. Also included are miscible formulations of solvate mixturessuch as a compound of the invention in combination with an acetone andethanol mixture. In a preferred embodiment, the solvate includes acompound of the invention in combination with about 20% ethanol andabout 80% acetone. Thus, the structural formulae include compoundshaving the indicated structure, including the hydrated as well as thenon-hydrated forms.

The term pharmaceutically acceptable prodrug as applied to the compoundsof the invention defines any pharmaceutically acceptable compound thatmay be converted under physiological conditions or by solvolysis to thespecified compound, to a pharmaceutically acceptable salt of suchcompound or to a compound that shares at least some of the antibacterialactivity of the specified compound (e.g. exhibiting activity againstClostridium difficile).

The term pharmaceutically acceptable metabolite as applied to thecompounds of the invention defines a pharmacologically active productproduced through metabolism in the body of the specified compound orsalt thereof.

Prodrugs and active metabolites of the compounds of the invention may beidentified using routine techniques known in the art (see for example,Bertolini et al., J. Med. Chem., 1997, 40, 2011-2016).

The term pharmaceutically acceptable complex as applied to the compoundsof the invention defines compounds or compositions in which the compoundof the invention forms a component part. Thus, the complexes of theinvention include derivatives in which the compound of the invention isphysically associated (e.g. by covalent or non-covalent bonding) toanother moiety or moieties. The term therefore includes multimeric formsof the compounds of the invention. Such multimers may be generated bylinking or placing multiple copies of a compound of the invention inclose proximity to each other (e.g. via a scaffolding or carriermoiety).

FIG. 6 is a generic structural formula showing the central core and ringpositions on a taurocholate molecule.

FIG. 7 is a more specific central core nucleus showing at least themajor positions on the taurocholate central core on which substituentsare placed within the scope of the present technology.

Specific chemical names for compounds within these groups ofconstituents include at least the following:

-   Tauracholate NH(CH₂)₂SO₃H-   2-[(3α,7α,12α-trihydroxy-24-oxo-5β-cholan-24-yl)amino]ethanesulfonic    acid-   NHCH₂SO₃H-   1-[(3α,7α,12α-trihydroxy-24-oxo-5β-cholan-24-yl)amino]methanesulfonic    acid-   NH(CH₃)₃SO₃H-   3-[(3α,7α,12α-trihydroxy-24-oxo-5β-cholan-24-yl)amino]propanesulfonic    acid-   NH(p-(C₆H₄))SO₃H-   4[(3α,7α,12α-trihydroxy-24-oxo-5β-cholan-24-yl)amino]benzenesulfonic    acid-   NH(o-(C₆H₄))SO₃H-   1-[(3α,7α,12α-Trihydroxy-24-oxo-5β-cholan-24-yl)amino]benzenesulfonic    acid-   NH(m-(C₆H₄))SO₃H-   3-[(3α,7α,12α-trihydroxy-24-oxo-5β-cholan-24-yl)amino]benzenesulfonic    acid-   NH(CH₂)₂SO₂H-   2-[(3α,7α,12α-trihydroxy-24-oxo-5β-cholan-24yl)amino]ethanesulfinic    acid-   Glycocholate NHCH₂CO₂H-   3α,7α,12α-Trihydroxy-5β-cholan-24-oic acid N-(carboxymethyl)amide OR    1-[(3α,7α,12α-trihydroxy-24-oxo-5β-cholan-24-yl)amino]methanecarboxylic    acid-   NH(CH₂)₂CO₂H-   2-[(3α,7α,12α-trihydroxy-24-oxo-5β-cholan-24-yl)amino]ethancarboxylic    acid-   NH(CH₂)₂CONH(CH₂)₂CO₂H-   6-[(3α,7α,12α-trihydroxy-24-oxo-5β-cholan-24-yl)amino]-(4-amino)hexanecarboxylic    acid-   NH(CH₂)₃CO₂H-   3-[(3α,7α12α-trihydroxy-24-oxo-5β-cholan-24-yl)amino]propanecarboxylic    acid-   NH(CH₂)₃CONH(CH₂)₃CO₂H-   8-[(3α,7α,12α-trihydroxy-24-oxo-5β-cholan-24-yl)amino]-(5-amido)ootanecarboxylic    acid-   O(CH₂)₂SO₃H-   2-[(3α,7α,12α-trihydroxy-5β-cholan-24-oyl)oxy]ethanesulfonic acid

EXAMPLES Materials and Methods Materials

Taurocholate and ammo acid analogs were purchased from Sigma-.AldrichCorporation (St Louis, Mo.), Steraloids (Newport, R.I.) or weresynthesized in the Abel-Santos laboratory. Reagents for synthesis werepurchased from Sigma-Aldrich Corporation (St. Louis, Mo.) or Alpha Aesar(Ward Hill, Mass.). Thin layer chromatography silica gel 60 F₂₅₄ waspurchased from EMD Chemicals (Gibbstown, N.J.). Silica gel for columnchromatography was purchased from Fisher Scientific (Pittsburg, Pa.).

Synthesis of 3-methoxy-7,12,-dihydroxytaurocholate (T09),3,7-dimethoxy-12-hydroxytauracholate (T10)

Methoxylated taurocholate analogs (T09 and T10) were prepared followingpublished procedures (Bandyopadhyay, P., V. Janout, L.-, Zhang, and S.L. Regen. 2001. Ion conductors derived from cholic acid and spermine:Importance of facial hydrophilicity on Na+ transport and membraneselectivity. J. Am. Chem. Soc. 123(31):7691-7696). Briefly, to asolution of taurocholate (1 equivalent) in dry 1,4-dioxane, methyliodide (50 equivalents) and sodium hydride (4 equivalents) were addedunder nitrogen. The reaction was heated to 40° C. for 48 hours withstirring. After the initial 48 hours sodium hydride (4 equivalents) wasadded daily to the reaction for four additional days. The reaction wasthen diluted with dichloromethane, washed with 1M HCl, and twice withwater. The organic layer was dried over anhydrous sodium sulfate and thesolvent was removed under reduced pressure. The resulting residue waspurified by silica gel column chromatography eluted with a step gradientfrom 100% dichloromethane to 60% dichloromethane/acetone. Two differentcompounds were obtained. ¹H-NMR and mass spectrometry showed that onecompound had a single methoxy group and the second compound had twomethoxy groups. The compounds were tentatively identified as3-methoxy-7,12-dihydroxytaurocholate (T09) and3,7-dimethoxy-12-hydroxytaurocholate (T10) as determined by published OHreactivity (Gargiulo, D., T. A. Blizzard, and K. Nakanishi. 1989.Synthesis of mosesin-4, a naturally occurring steroid saponin with sharkrepellent activity, and its analog 7-β-galactosyl ethyl cholate.Tetrahedron, 45(17):5423-5432, Iida, T. and F. C. Chang. 1982. Potentialbile acid metabolites. 6. Stereoisomeric 3,7-dihydroxy-5β-cholanicacids. J. Org. Chem. 47(15)2966-2972.).

Synthesis of CAAMSA (T11), CAAPSA (T12), CApSA (T13), CAoSA (T14), CAmSA(T15), hypotaurocholate (T16), CAAPA (T18), CA2APA (T19), CAABA (T20),and CA2ABA (T21)

Taurocholate analogs T11-T16 and T18-T21 were prepared followingpublished procedures (Dayal, B., K. R. Rapole, C. Patel, B. N. Pramanik,S. Shefer, G. S. Tint, and G. Salen. 1995, Microwave-induced rapidsynthesis of sarcosine conjugated bile acids. Bioorg. Med. Chem. Lett.5(12):1301-1306. Tserng, K. Y., D. L. Hadley, and P. D. Klein. 1977. Animproved procedure for the synthesis of glycine and taurine conjugatesof bile acids. J. Lipid Res. 18(3):404-407.). Briefly, cholic acid (1equivalent) was activated with 1.4 equivalents ofN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and 1.3equivalents N-methylmorpholine (NMO) in dimethylformamide (DMF). Afterstirring for 5 minutes, 1.2 equivalents of the appropriate aminosulfonic acid or amino acid were added. The reaction was heated to 90°C. for 40 min and then cooled to room temperature. The solution waspoured into 100 ml of ice-cold diethyl ether resulting in a precipitate.The ether suspension was kept at 4° C. overnight. The ether layer wasdecanted and the resinous residue was dissolved in 25 ml 0.2 N NaOH/MeOHand poured into 100 ml cold, diethyl ether. The ether solution was keptat 4° C. for at least 2 hours and the resulting precipitate was filteredand washed with diethyl ether. Unnecessary, the product wasrecrystallized by dissolving in hot ethanol to saturation followed bythe addition of ethyl acetate until a precipitate appeared. The solutionwas kept at −20° C. for 2 hours to allow complete precipitation and thenfiltered to retrieve product. The precipitated residue was furtherpurified by silica gel column chromatography eluted with a step gradientfrom 100% dichloromethane to 100% ethanol. CA2APA (T19) and CA2ABA (T21)were obtained as side products of the synthesis of CAAPA (T18), andCAABA (T20), respectively. Compound structures were verified by ¹ H-NMR,FT-IR, and mass spectrometry.

Synthesis of CAHESA (T22)

Conjugation of cholate to the sulfonic acid alkyl linker by an ester wasprepared following established protocols for Fischer esterification(e.g., Fischer, E. and A. Speier. 1895. Darstellung der Ester. Ber.28(3):3252-3258. Indu, B., W. R. Ernst, and L. T. Gelbaum. 1993.Methanol-formic acid esterification equilibrium in sulfuric acidsolutions: Influence of sodium salts. Ind. Eng. Chem. Res.32(5):981-985.). Briefly, to a solution of cholate (1 equivalent) andhydroxy ethane sulfonic acid (4 equivalents), concentrated sulfuric acidwas added dropwise and refluxed for one hour. The reaction was pouredinto cold diethyl ether and a precipitate formed immediately. Thediethyl ether suspension was left overnight at 4° C. The precipitate wasfiltered, dissolved in 0.2 N NaOH/MeOH, precipitated a second time indiethyl ether, and kept at 4° C. for at least 2 hours. The crudeprecipitate was filtered and purified by silica gel columnchromatography eluted by step gradient from 100% dichloromethane to 30%dichloromethane/EtOH. The compound structure was verified by ¹H-NMR,FT-IR, and mass spectrometry.

Bacterial Strains and Spore Preparation

Clostridium difficile strain 630 (ATCC BAA-1382) was obtained from theAmerican Type Culture Collection (ATCC). C. difficile cells were platedin BHIS agar supplemented with 1% yeast extract, 0.1% L-cysteine HCl and0.05% sodium taurocholate to yield single cell clones. Single C.difficile colonies were grown in BHIS broth and spread plated onto agarto obtain bacterial lawns. Plates were incubated for 5 days at 37° C. inan anaerobic environment (5% CO₂, 10% H₂, and 80% N₂). The resultingbacterial lawns were collected by flooding with ice-cold deionizedwater. Spores were pelleted by centrifugation and resuspended in freshdeionized water. After two washing steps, spores were separated fromvegetative and partially sporulated forms by centrifugation through a20%-50% HistoDenz gradient. The spore pellet was washed five times withwater, resuspended in sodium thioglycollate (0.5 g/liter) and stored at4° C.

Activation of C. Difficile Spore Germination by Amino Acids andTaurocholate analog

Spores were diluted in germination buffer (100 mM sodium phosphatebuffer (pH 6.0) containing 5 mg/ml sodium bicarbonate) to an OD₅₈₀ of1.0. To test for taurocholate agonists of spore germination, sporesuspensions were individually supplemented with 6 mM of thecorresponding taurocholate analogs and 12 mM glyrine. To test for aminoacid agonists of spore germination, spore suspensions were supplementedwith 12 mM of the corresponding amino acid analogs and 6 mMtaurocholate. Spore germination was evaluated based on decrease in OD₅₈₀at 30° C. each minute for 90 min. Germination rates were set to 100% forC. difficile spores germinated with 6 mM sodium taurocholate and 12 mMglycine. Percentage germination for all analogs was calculated as thefraction of germination rate compared to these conditions.

Inhibition of C. Difficile Spore Germination by Amino Acids andTaurocholate Analogs

To test for antagonists of spore germination, more aliquots wereindividually supplemented with varying concentration of taurocholateanalogs or amino acid analogs. Spore suspensions were incubated for 15min at mom temperature while monitoring OD₅₈₀. If no germination wasdetected, taurocholate and glycine were added to 6 and 12 mM finalconcentrations, respectively. Relative OD₅₈₀ values were obtained everyminute for 90 minutes after germinant addition and were plotted againstthe logarithm of inhibitor concentrations. As expected, germinationdecreased in the presence of active germination inhibitors. Theresulting data were fitted using the four parameter logistic functionSigmaPlot v.9 software to obtain IC₅₀ values (Rodbard, D. and Y.Feldman. 1978. Kinetics of two-site immunoradiometric (‘sandwich’)assays. I. Mathematical models for simulation, optimization, and curvefitting. Mol. Immunol. 15(2):71-76. Rodbard, D. and S. W. McClean. 1977.Automated computer analysis for enzyme multiplied immunologicaltechniques. Clin. Chem. 23(1):112-115).

C. Difficile Spore Germination in BHIS Media

To test for germination in complex media, spores were resuspended inbrain heart infusion broth supplemented with 5 mg/ml yeast extract and0.1% L-cysteine (BHIS) alone and with combinations of taurocholate,chenodwxycholate, glycocholate, glycine, L-arginine, andL-phenylalanine. Bile salts were added to a final concentration of 6 mMand amino acids were added at a final concentration of 12 mM. RelativeOD₅₈₀ so values were obtained every minute for 90 minutes area germinantaddition.

C. difficile has been shown to germinate in the presence of glycine andtaurocholate (35). The question that has remained unanswered is howglycine and taurocholate interact with the putative binding sites. Inthe current work, we have tested 30 amino acid analogs and 22taurocholate analogs as activators (FIG. 1A) or inhibitors of C.difficile spore germination (FIG. 1B). Structure-activity relationshipanalysis of germinant analogs allows a better understanding of themicroenvironment of the C. difficile germination machinery. Activatorsof the germination pathway identify which functional groups areessential for binding and activation of the C. difficile germinationmachinery. On the other hand, inhibiting agents provide structuraldetails about functional groups that allow only binding. Inhibitionassays serve as an indirect method to map physiochemical configurationsin receptor binding sites. Competitive inhibitors most likely bind tothe same site as the cognate germinant. Strong competitive inhibitorswill complement the germinant binding site shape, size, hydrophobicity,and hydrogen bonding pattern. Inactive compounds yield information onfunctional groups that interfere with germinant binding. As expected,changing glycine and taurocholate functional groups affected thegermination of C. difficile spores.

Glycine (A01) has a methylene bridge that separates the carboxylic andamine groups and is the simplest of the 20 common amino acids. To finddeterminants required for glycine recognition, we individuallysupplemented taurocholate-treated spores with 30 different glycinanalogs (FIG. 2). Each of the glycine analogs differ from the parentcompound by a single modification, either in the length of the alkylchain, substitutions to the amino group, or changes in the carboxylategroup. β-Alanine (A02) and γ-aminobutyric acid (A03) have an ethyleneand a propylene bridge between the amino and carboxylate group,respectively. These changes progressively increase the distance betweenthe amino and carboxylate groups. β-Alanine (A02) and γ-aminobutyricacid (A03) are as effective as glycine as co-germinants of C. difficilespores (FIG. 3). Thus, lengthening the chain between the amino andcarboxylate functional groups does not interfere with recognition by theputative glycine germination receptor.

Aminomethylphosphonic acid (A04) is a glycine analog where thecarboxylate has been changed to a phosphonate. This substitutionexchanges a carbon atom for phosphorus while retaining the negativecharge. Aminomethylphosphonic acid (A04) significantly decreased C.difficile spore germination (FIG. 3). Furthermore, methylation of thecarboxylate in glycine methyl ester (A05) resulted in germination ratesof less than 10% compared to glycine-triggered germination. Any othermodification of the carboxylate (glycine ethyl ester (A06), glycinamide(A07), glycine hydroxamate (A08)) resulted in compounds that were unableto activate or inhibit C. difficile spore germination data not shown).The sum of these data suggests that there is a specific requirement fora carboxylate functional group for recognition by the glycinegermination receptor to activate germination.

The distance between the amino and carboxylate of diglycine (A09) issimilar to γ-aminobutyric acid (A03). However, whereas γ-aminobutyricacid (A03) is a good agonist for C. difficile spore germination,diglycine (A09) has no effect (data not shown). Thus the addition of aninternal amide must interfere with compound binding Possibly there is arequirement for a more hydrophobic linker between the two functionalgroup ends. Glycine anhydride (A10) is the result of the dehydration ofdiglycine, forming a cyclic diamide. Without a free amine orcarboxylate, this compound is unable to activate or inhibit sporegermination (data not shown). The rigidity and bulkiness of this analogmay prevent interaction with the glycine binding site.

Alkylation (sarcosine (A11), N,N-dimethylglycine(A12), betaine (A13)),acetylation (N-acetylglycine (A14)), or other modification(nitrilotriacetic acid (A15)) to the amino group of glycine resulted incompounds that can neither activate nor inhibit C. difficile sporegermination (data not shown). This suggests that activation of C.difficile spores by glycine has a requirement for a free primary aminogroup regardless of an unmodified carboxylate group. To test the effectof side-chain substitution in amino acid recognition by the C. difficilegermination machinery, we exposed taurocholate-treated C. difficilespores to other amino acid analogs (FIG. 2). L-alanine (A16) has beenshown to act as germinant and/or co-germinant in other sporulatingbacteria (Abel-Santos. E. and T. Dodatko. 2007. Differential nucleosiderecognition during Bacillus cereus 569 (ATCC 10876) spore germination.New J. Chem. 31(5):748-755). The stereoisomer, D-alanine (A17), has beenshown to inhibit alanine-mediated germination in Bacillus (Preston, R.A. and H. A. Douthit, 1988, Functional relationships between L- andD-alanine, inosine and NH4Cl during germination of spores of Bacilluscereus. T. J. Gen. Microbiol. 134(11):3001-3010.). In C. difficileL-alanine (A16) was as efficient at triggering germination as glycine(A01) (FIG. 3). Interestingly, D-alanine (A17) was unable to inhibitgermination of spores treated with L-alanine and taurocholate (data notshown). D-alanine was also inactive as an agonist for C. difficile sporegermination (data not shown). This implies that stereochemistry isimportant for recognition and binding of amino acids.

To determine whether amino acid analogs with longer, linear alkyl sidechains were able to activate C. difficile spore germination, we exposedtauracholate-treated spores to L-2-aminobutyric acid (A18) andL-norvaline (A19). Both of these amino acid analogs were able toactivate germination to levels similar to glycine (A01), L-valine (A20)is a branched isomer of L-norvaline (A19) and has similar chemical andphysical properties. However, this slight difference in structurereduced C. difficile spore germination by more than 90% compared toL-norvaline (FIG. 3). Similarly, L-isoleucine (A21) and L-leucine (A22)are poor germinants of C. difficile spores compared to L-norvaline(A19). The data suggests that branched alkyl side chains are unable tobe recognized by the putative amino acid germination receptor in C.difficile.

L-cysteine (A23) is an amino acid with a methanethiol side chain.L-serine (A24), on the other hand, is an L-cysteine analog where thethat group is substituted for a hydroxyl group. Interestingly, whereasL-cysteine (A23) is a good germinant of C. difficile spores (FIG. 3),the more polar L-serine (A24) is almost inactive. The germinationactivity of L-cysteine (A23) is not due solely to hydrophobicity sincethe more hydrophobic L-methionine (A25) is a poor germinant compared toL-cysteine (A23). This suggests that C. difficile spores recognize thethiol group specifically as a determinant for germination.

Surprisingly, L-phenylalanine (A26), with a bulky aromatic side chain,is as effective as glycine as a germinant of C. difficile spores (FIG.3). We would expect that since branched amino acids are inactive, thebulkier side chain of L-phenylalanine would also be restricted from thebinding site. We cannot completely rule out that the putative glycinereceptor is able to accommodate phenyl, but not branched alkyl sidechains. However, we find that possibility unlikely due to their relativesize compared to glycine. Hence, we postulate that aromatic amino acidsare recognized by a separate binding site in the C. difficile spore.Indeed, other Bacillus and Clostridium are able to recognizestructurally different germinants by encoding multiple receptors (e.g.,Dodatko, T. M. Akoachere, N. Jimenez, Z. Alvarez, and E. Abel-Santos2010.

Dissecting interactions between nucleosides and germination receptors inBacillus cereus 569 spores. Microbiology. 156(4)1244-1255. Foster, S. J.and K. Johnstone. 1990. Pulling the trigger. The mechanism of bacterialspore germination. Mol. Microbiol. 4(1)137-141.).

L-arginine (A27) is also a strong co-germinant for C. difficile spores(FIG. 3). Although L-arginine has a linear alkyl chain, it also containsa positively charged, branched guanidinium group. Similarly, L-lysine(A28) is linear with a positively charged amino side chain. Yet L-lysine(A28) is a weak germinant compared to L-arginine, L-histidine (A29)contains an aromatic side chain like L-phenylatanine (A26) but ispositively charged like L-arginine's (A27) side chain. However, unlikeL-phenylatanine (A26) or L-arginine (A27), L-histidine (A29) could notefficiently activate C. difficile spore germination. L-aspartic acid(A30) has a short acidic side chain and was similarly unable to affectC. difficile spore germination (data not shown). Thus, L-arginine (A27)was the only polar and/or charged amino acid tested that. was able toinduce C. difficile spore germination. Since L-arginine (A27) hasphysicochemical properties that are very different from the other aminoacids able to activate C. difficile spore germination, it suggests thereis a specific recognition site for L-arginine binding.

Earlier studies (Sorg, J. A. and A. L. Sonenshein. 2008. Bile salts andglycine as cogerminants for Clostridium difficile spores. J. Bacterial.190(7):2505-2512.) reported that only glycine was able to trigger C.difficile spore germination in the presence of taurocholate. This studyreportedly used three combinations of defined media to narrow downactive germination stimulators. Although glycine was supplemented withonly seven amino acids, phenylalanine and arginine were in mixturesupplemented with 15 other amino acids. The presence of multiple weakgen/imams in the defined media containing phenylalanine and argininecould mask their ability to stimulate germination. Furthermore, in thoseexperiments L-phenylalanine and L-arginine were supplemented at lowerconcentrations (1.21 mM and 1.15 mM, respectively) than used for thecurrent experiments (12 mM).

Glycine, L-arginine, and L-phenylalanine individually or in pairs do nottrigger C. difficile spore germination in the absence of taurocholate(data not shown). However, a cocktail of L-phenylalanine, L-arginine,and glycine (all at 12 mM) was able to effectively trigger C. difficilespore germination in the absence of taurocholate (FIG. 4).Chenodeoxycholate (6 mM) is not able to inhibit spore germination of C.difficile spores treated with amino acids only. Other Clostridia havebeen shown to use amino acids alone as germination signals (Ramirez, N.and E. Abel-Santos 2010. Requirements for germination of Clostridiumsordellii spores in vitro. J. Bacteria 192(2):418-425.).

When C. difficile spores are resuspended in BHIS media, germination isvery slow even though BHIS contains a complex amino acid mixture thatincludes glycine, L-arginine, and L-phenylalanine (FIG. 4). It ispossible that BHIS contains amino acids that are weak germinants andwill compete with glycine, L-phenylalanine, and L-arginine far binding.Binding of these alternative substrates will cause a fraction of thespore population to germinate at a slower rate. We have seen similarbehavior in the germination of C. sordellii spores (27). Interestingly,supplementing BHIS with taurocholate (FIG. 5, T01) augmented C.difficile spore germination (FIG. 4). The taurocholate-enhanced sporegermination in BHIS was inhibited by chenodeoxycholate. Thus, it seemsthat amino acid-only C. difficile spore germination occurs only when alimited number of amino acids are present and is disfavored with complexamino acid mixtures.

All glycine analogs and amino acids were further tested for theirability to inhibit C. difficile spore germination. Spores were treatedwith each analog or amino acid in the presence of taurocholate andglycine. However, no individual amino acid analog inhibited C. difficilespore germination (data not shown).

Taurocholate (T01) is a natural bile salt that has hydroxyl groups atposition 3, 7, and 12 of the cholate skeleton. All three hydroxyl groupsare in the alpha-configuration. Taurocholate also has a side chainconsisting of taurine attached to the cholate skeleton by an amide bond.Taurocholate activates C. difficile spore germination with an EC₅₀ of15.9 mM. Taurocholate analogs were tested for their ability to activategermination in the presence of glycine and for their ability to inhibitgermination in the presence of taurocholate and glycine.

To understand the importance of hydroxyl groups on the cholate backboneof taurocholate, analogs T02-T08 (FIG. 5) were tested as agonists andantagonists of C. difficile spore germination. These analogs differ fromtaurocholate (T01) in the number, placement, or stereochemistry of thehydroxyl groups. Tamodeoxycholate (T02) lacks only the hydroxyl group atthe 7-position. This change was sufficient to reduce germination by morethan 70% (Table 1). Meanwhile, taurochenodeoxycholate (T03) is onlymissing the hydroxyl at position 12 and was only able to inducegermination to 10% of taurocholate. Tauroursodeoxycholate (T04) is anisomer of taurochenodeoxycholate (T03) where the 7-hydroxyl is in thebeta-configuration. The alteration of stereochemistry of this onehydroxyl group further decreased germination activity from 10% fortaurochenodeoxycholate (T03) to 3% for tauroursodeoxycholate (T04)(Table 1).

As expected, taurolithocholate (T05) and taurocholanate (T06) that lackhydroxyls at positions 7, 12 and 3, 7, 12, respectively are unable toactivate germination. Taurohycholate (T07) and taurohyodeoxycholate(T08) are isomers of taurocholate (T01) and taurodeoxycholate (T02),respectively where the 12-hydroxyl groups were moved to the 6-position.Neither of these compounds is able to significantly activate germinationof C difficile spores. The sum of these data suggests that both the 7and 12 α-hydroxyls of taurocholate are important determinants forbinding and activation of C. difficile spores.

The 3-hydroxyl position of the cholate molecule is more nucleophilicthan the other two hydroxyls. Similarly, the 7-hydoxyl is more reactivethan the 12-hydroxyl (13, 15). Thus, methylation of taurocholate yieldedtwo compounds that we putatively identified as3-methexy-7,12-dihydroxytaurocholate (T09) and3,7-dimethoxy-12-hydroxytaurocholate (T10). interestingly,3-methoxy-7,12-dihydroxytaurocholate (T09) neither induces nor inhibitsC. difficile spore germination. As expected,3,7-dimethoxy-12-hydroxytaurocholate (T10) was also inactive. Thissuggests that the 3-hydroxyl hydrogen bond donating ability is essentialfor recognition of taurocholate as a germinant C. difficile spores.

To determine if analogs differing in the number location, orstereochemistry of the hydroxyl groups can inhibit taurocholate mediatedgermination, C. difficile spores were treated with taurocholate (T01),glycine (A01), and compounds T02-T10 (Table 1). Onlytaurochenodeoxycholate (T03), tauroursodeoxycholate (T04) andtaurohyodeoxycholate (T08) showed germination inhibitory properties. Allthree inhibitors have the common feature of lacking the 12-hydroxylgroup. Since the 12-hydroxyl group was necessary for triggering sporegermination, these results suggest that this hydroxyl is necessary foractivation of germination but not for binding of taurocholate to theputative C. difficile germination receptor. The inhibitory compoundsalso have hydroxyl groups at either the 6- or 7-positions (but not both)indicating that having one (but not two) hydroxyl in the B-ring isimportant for inhibition of taurocholate-mediated germination of C.difficile spores.

To determine taurine side chain functional groups responsible forrecognition by the C. difficile germination machinery, analogs T11-T22were tested for the ability to induce spore germination in the presenceof glycine (FIG. 5). All these compounds differ from taurocholate in thestructure of the side chain and retain the cholate skeleton withhydroxyl groups at the 3, 7, and 12 positions.

CAAMSA (T11) is a taurocholate analog where the alkyl linker between thesulfonate and the amide was shortened by one methylene. CAAPSA (T12), onthe other hand is a taurocholate analog where the linker was lengthenedby one methylene (FIG. 5). The results show that spores treated withCAAMSA (T11) germinate to levels comparable with spores treated withtaurocholate (T01) (Table 2), in contrast the longer alkyl chain, CAAPSA(T12), does not activate germination. To further analyze the necessityof the alkyl linker on germination, we synthesized three analogscontaining a benzene ring in place of the ethylene linker of taurine(FIG. 5). These analogs differed in the position of the sulfonate, para(CApSA, T13), ortho (CAoSA, T14), or meta (CAmSA, T15), with respect tothe amino group in the benzene ring. These three taurocholate analogswere unable to activate C. difficile spore germination. This suggeststhat C. difficile spores are activated by taurocholate analogs withshorter, but not longer or bulkier linkers.

Hypotaurocholate (T16) differs from taurocholate by a substitution ofthe sulfonate for a sulfinate (FIG. 5). This analog was unable toactivate C. difficile spore germination. Interestingly, glycocholate(T17) is a CAAMSA (T11) analog where the sulfonate has been substitutedfor a carboxylate. In our hands, glycocholate (T17), like CAAMSA (T11)is able to significantly activate C. difficile spore germination inbuffer (Table 2). A previous report had determined that glycocholate(T17) is not a germinant for C. difficile spores in BHIS media (35).Indeed, when we tested glycocholate (T17) in BHIS media, C. difficilespore germination rate dropped almost 10-fold. However, addition ofglycine to BHIS media partially restored glycocholate-mediatedgermination (data not shown). Hence, compound mixtures in BHIS mediaseem intrinsically inhibit C. difficile spore germination.

CAAPA (T18) is a carboxylated analog of taurocholate (T01).Interestingly, whereas taurocholate (T01), CAAMSA (T11) and glycocholate(T17) are able to activate C. difficile spore germination, CAAPA (T18)is inactive. Similarly, CA2APA (T19), CAABA (T20), and CA2ABA (T21) withlonger side chains are also inactive. These results suggest thatalthough a carboxylate is able to partially substitute for sulfonate, itis not optimal for activation of C. difficile spore germination.

CAHESA (T22) is a taurocholate analog where the amide group issubstituted for an ester. CAHESA (T22) was unable to trigger germinationin C. difficile spores (Table 2). This suggests that the hydrogen bondability of the amide group is necessary for C. difficile sporegermination.

To determine whether taurocholate analogs with modified side chains(T11-T22) (FIG. 5) can inhibit C. difficile spore germination, weanalyzed the effect of these analogs on C. difficile spores treated withtaurocholate (T01) and glycine (A01). Taurocholate analogs with one lesscarbon (T11) or one more carbon (T12) in the taurine side chain wereunable to inhibit germination (Table 2). Interestingly, while theaddition of a benzene ring in the linker with the sulfonate in the para(CApSA, T13) or the ortho (CAoSA, T14) position does not inhibit sporegermination, having the sulfonate in the meta position (CAmSA, T15)results in strong inhibition of C. difficile spore germination. CAmSA(T15) has an IC₅₀ of 58.3 μM and is the strongest inhibitor reported sofar. The conversion of the sulfonate functional group to a sulfinate(T16) resulted in a compound with slight inhibitory activity with anIC₅₀ of 640 μM. Interestingly, compounds with longer alkyl linkersfollowed by a carboxylate (T20-T21) are able to inhibit C. difficilespore germination whereas shorter carbo end groups (T17-T19) areinactive. CAABA (T20) has an IC₅₀ of 762 μM whereas the longer sidechain, CA2ABA (T21), containing two amide groups has an IC₅₀ ofapproximately 3,000 μM. Replacement of the amide group of taurocholatefor an ester (T22) results in a compound with slight inhibitoryactivity, IC₅₀ of 1,322 μM.

FIG. 1. Germination kinetic graphs showing agonistic and antagonisticbehavior of molecules with C. difficile spores. (A) Activation ofgermination C. difficile spores were treated with a 34 fixedconcentration of taurocholate (6 mM) and glycine was added at 0 mM (∘),8 mM (), 10 mM (□), 12 mM (▪), and 14 mM (Δ) final concentrations. Forclarity, data is shown at five minutes intervals and only for fiveglycine concentrations. (B) Inhibition of germination; C. difficilespores were incubated with 0 mM (∘), 0.0005 mM (), 0.001 mM (□), 0.075mM (▪) and 7.5 mM (Δ) concentrations of CAmSA (T15) and supplementedwith taurocholate (6 mM) and glycine (12 mM). For clarity, data is shownat five minutes intervals and only for five CAmSA (T15) concentrations.Although data was collected for 90 minutes, only 75 minutes are shown inboth graphs for clarity.

FIG. 2. Amino acids assessed for activation or inhibition ofglycine-mediated germination in C. difficile spores. Glycine (A01),β-Amine (A02), γ-aminobutyric acid (A03), aminomethylphosphonic acid(A04), glycine methyl ester (A05), glycine ethyl ester (A06),glycinamide (A07), glycine hydroxamate (A08), diglycine (A09), glycineanhydride (A10), sarcosine (A11), N,N-dimethylglycine (A12),betaine(A13), N-acetylglycine (A14), nitrilotriacetic acid (A15),L-alanine (A16), D-alanine (A17), L-2-aminobutyric acid (A18),L-norvaline (A19), L-valine (A20), L-isoleucine (A21), L-leucine (A22),L-cysteine (A23), L-senile (A24), L-methionine (A25), L-phenylalanine(A26), L-arginine (A27), L-lysine (A28), L-histidine (A29), L-asparticacid (A30).

FIG. 3. Comparison of amino acids as agonists of C. difficile sporegermination. Spores were treated with taurocholate (6 mM) and amino acidanalog at 12 mM. Germination was determined by the decrease in OD₅₈₀ for90 minutes at 30° C. Percent germination for each analog was calculatedbased on glycine/taurocholate germination as 100%.

FIG. 4. Germination kinetic graph showing behavior of C. difficilespores and germinants in buffer and complex media. C. difficile sporeswere resuspended in germination buffer and treated with L-phenylalanine,L-arginine, and glycine (each at 12 mM) (∘), or L-phenylaline.L-arginine, glycine (each at 12 mM) and chenodeoxycholate (6 mM) ().Purified spores were also suspended in BHIS medium (□). BHISsupplemented with 12 mM taurocholate (▴), or BHIS supplemented with 12mM taurocholate and 12 mM chenodeoxycholate (Δ). For clarity, data isshown at five minutes intervals and only for 75 minutes.

FIG. 5. Taurocholate analogs assessed for activation or inhibition oftaurocholate-mediated germination in C. difficile spores. Taurocholate(T01), taurodeoxycholate (T02), taurochenodeoxycholate (T03),tauroursodeoxycholate (T04), taurolithocholate (T05), taurocholanate(T06), taurohycholate (T07), taurohyodeoxycholate (T08),3-methoxy-7,12-dihydroxylaurocholate (T09),3,7-dimethoxy-12-hydroxytaurocholate (T10), CAAMSA (T11), CAAPSA (T12),CApSA (T13). CAoSA (T14), CAmSA (T15), hypotaurocholate (T16),glycocholate (T17), CAAPA (T18), CA2APA (T19), CAABA (T20), CA2ABA(T21), CAHESA (T22)

In these examples, we used taurocholate and glycine analogs to betterunderstand how the C. difficile germination machinery recognizes itsgerminants. Chemical probes can reveal chemical, physical, and spatialrequirements of the germination receptor binding site. This presentstudy has shown that C. difficile germination machinery recognizes atnumber of amino acid side chains and that the putative glycine receptorrequires both a free carboxylate and a free amino group to recognizeglycine, but the binding site is flexible enough to accommodate longerseparations between the two functional groups. Alkyl amino acid sidechains seem to be recognized in a narrow hydrophobic groove that allowsthe binding of linear chains but excludes branched isomers. We have seena similar branched chain restriction in the recognition of inosineanalogs by B. cereus spores (9). Those size and polarity restrictionsalso suggest the existence of separate binding sites forL-phenylalanine, L-arginine and possibly L-cysteine.

We suggest that the binding region for L-alanine in C. difficile isdivergent enough from the L-alanine binding site in Bacillus to impedethe binding and inhibition by D-alanine. Because none of the amino acidanalogs was able to compete with glycine to inhibit C. difficile sporegermination, the functional groups in the amino acid moieties are neededfor both binding and activation of the putative amino acid germinationreceptor.

The putative taurocholate binding sites of C. difficile spores were lessflexible in compounds allowed to bind and activate germination. Hydroxylgroups at position 3 and 12 seem to be required for both binding andactivation of C. difficile spore germination, in contrast, hydroxylgroups in the B-ring appear to be important only for binding. Hence, thedata implies there is a requirement for hydrogen bonding with hydroxylsat specific locations and configurations in the C. difficile germinationbinding pocket.

Recognition of the taurine side chain seems to be even more restricted.Even small changes in linker length and rigidity, amide bond, oroxidation state of the sulfonate group had a large effect on C.difficile spore germination. Although the sulfonate group is optimal forspore germination activation, it can be partially substituted with acarboxylate as long as the alkyl chain is short. This data suggests thatthe binding site for taurocholate recognizes the taurine side chainselectively.

In contrast to agonist specificity, the putative taurocholate bindingreceptor was more flexible in regards to antagonist binding. Themeta-sulfonic benzene derivative CAmSA (T15), is active atconcentrations approximately 275-fold lower than taurocholate and isalmost 4 times more active than the natural inhibitor chenodeoxycholate.The benzene ring is a rigid functional group with little free rotation.We speculate the sulfonate in the meta position is able to fit tightlyinto the sulfonate binding pocket but the overall receptor does notrecognize the benzene ring to trigger germination. This is furtherconfirmed by the inactivity of the who and para isomers that wouldspatially place the sulfonate in different locations. The rigidity andpositioning of the m-sulfonate probably provides an entropic advantageover alkyl sulfonates. It is possible that longer alkyl side chains aretoo flexible to allow the sulfonate moiety to bind efficiently to theputative taurocholate. The discovery of CAmSA (T15) and its stronginhibitory effect has revealed a new path to designing compounds for CDIprophylaxis.

Interestingly, BHIS media seem to have intrinsic inhibitory activityagainst amino acid (but not taurocholate) activation of C. difficilespore germination. Since biological media is a better representation ofpotential metabolites present in the host, it indicates thattaurocholate-mediated germination is the prefer pathway for humaninfection.

In conclusion, the putative taurocholate glycine receptors in C.difficile recognize multiple functional groups in their respectivegerminants. Hence, even subtle changes in the germinant structure can bedetrimental to the binding ability of the germination machinery of C.difficile spores.

Recent Findings Regarding the Prophylactic Activity or CamSA onClostridium Difficile Infection

i. Testing for CamSA Toxicity in Mice.

Before we embarked in establishing a CDI animal model, we tested furCamSA toxicity in mice following a modification of the Fixed DoseProcedure (FDP). To determine acute toxicity of CamSA, groups of 5animals were treated for 3 consecutive days with single gavage doses of50 mg/Kg CamSA in DMSO. Animals were observed for 5 days for signs oftoxicity (e.g. weight loss, diarrhea, hunch back, lethargy. Treatedanimals showed no signs of toxicity and were indistinguishable fromanimals treated with neat DMSO or neat water. Since no toxicity wasobserved, a second group of animals was dosed at 300 mg/Kg CamSA. Asabove, no signs of toxicity were apparent at this higher dose.

ii. Preparation of C. difficile Spores for CDI challenge:

A literature review showed that different conditions for C. difficilesporulation have been used to prepare spores for murine CDI challenge.More importantly, the published work establishing a mouse CDI model didnot report spore purity. Furthermore, vegetative C. difficile cells wereused to create a moose relapse model for CDI, but no sporequantification was reported either. In our experience, growth conditionscan affect the extent of sporulation and impure spore preparations canaffect germination kinetics (1). Because of these issues, our laboratoryhas established details protocols to purify C. difficile spores tohomogeneity.

To determine the effect of spore purity on CDI establishment, weprepared C. difficile spores using methods reported in two mice CDImodels. We also tested C. difficile spore preparations that werepurified following procedures from our laboratory. Furthermore, wetested two different C. difficile strains. C. difficile strain VPI 10463has been used to induce mouse CDI, but sporulates poorly. C. difficilestain 630 produces abundant spores, but has not been tested in the CDImouse model. The amount of spores produced under each condition wasmeasured by microscopy observation of Schaeffer-Fulton stained aliquots.As expected, strain VPI 10463 yielded low amounts of spores, whilestrain 630 yielded large quantities of spores. Using sporulationconditions reported for the mouse CDI model, yielded preparation thatconsisted almost exclusively of vegetative cells (<5% spores).

iii. Refining the Mouse CDI Model:

Each spore preparation above was used to infect groups of 5 mice withCFU of spores following published procedures. Under these conditions,purified spores from both the VPI 10463 and 630 strains caused CDIsymptoms in mice (diarrhea, weight loss, wet-tail, lethargy). Incontrast, mice challenged with C. difficile vegetative cells did notdevelop CDI. Similar results were observed when mice the initial dose ofC. difficile spores or vegetative cells were increased to 10⁸ CFU. Fromthese experiments we established that the best conditions to establishCDI involves highly purified C. difficile strain 630 spores.

iv. Testing CamSA as CDI Prophylaxis:

To determine if CamSA protects mice from CDI, groups of five mice weretreated with 5, 25, 50 or 300 rag/kg of either CamSA (a germinationinhibitor) or taurocholate (a germination activator) in DMSO. A thirdgroup was given neat DMSO. Each group was then infected with 10⁸ CFU ofpurified C. difficile strain 630 spores.

Each group was given repeated doses of CamSA, taurocholate or DMSO at 24and 48 hours post-challenge. All animals were weighted twice a day andmonitored for CDI symptoms. As above, DMSO-treated animals developed CDIin the first 48 hours after challenge. Similarly, alltaurocholate-treated animals developed CDI. In contrast, animals treatedwith 50 or 300 mg/Kg CamSA did not develop CDI and wereundistinguishable from non-infected animals. Animals treated with 5 or25 mg/Kg CamSA developed CDI, but disease onset and severity were lesspronounced than either DMSO or taurocholate treated animals. Thus,CamSA, but not taurocholate, is able to prevent CDI in mice (Table 1).

v. Testing CamSA Dosage Timing in CDI Prevention:

To determine the latest time point for effective CamSA prophylaxis,antibiotic treated mice were infected with 10⁸ CPU of purified C.difficile strain 630 spores. Mice were divided into four groups. GroupsI, II, III, and IV were given a 300 mg/Kg dose of CamSA at 0, 3, 6, and12 hours after spore challenge, respectively (Table 2). A second dose ofCamSA was administered 24 hours after the first dose. All animals wereweighted twice a day and monitored for CDI symptoms. Animals treatedwith CamSA at 0, 3, and 6 hours post-infection did not develop any CDIsymptoms and were undistinguishable from control animals. In contrast,all animals treated with CamSA at 12 hours post-infection developed CDIsymptoms. Although the onset of the disease was delayed by 24 hourscompared to DMSO-treated animals, the severity of the disease was notabated. This data shows that there is at least a 6 hour window for CamSAto be effective In CDI prevention. Even more, spore germination is arequired first step that occurs prior to the appearance of CDI symptoms.Thus, the protective effect of CamSA can be used to estimate that C.difficile spore germination occurs between six hours and 12 hours afterC. difficile spore ingestion.

TABLE 1 Testing CamSA as CDI prophylactic Average weight Animals withCDI change 3 days Treatment symptoms post challenge DMSO 4/4 −12%Taurocholate 3/3 −18% CamSA (300 mg/Kg) 0/5 +06% CamSA (50 mg/Kg) 0/5+05% CamSA (25 mg/Kg) 5/5 −05% CamSA (5 mg/Kg) 5/5  −8%

TABLE 2 Testing CamSA dosage timing First CamSA dose after Animals withCDI Group challenge (h) symptoms I 0 0/5 II 3 0/5 III 6 0/5 IV 12 5/5

In FIG. 7,

-   -   ^(a) C. difficile spores were individually treated with 6 mM        glycine and 12 mM taurocholate analogs T11-T22. Percent        germination was calculated based on taurocholate/glycine        germination as 100%. Standard deviations are shown in        parentheses.    -   ^(b) C. difficile spores were incubated with various        concentrations of taurocholate analogs for 15 minutes prior to        addition of 6 mM taurocholate and 12 mM glycine. IC₅₀ was        calculated by plotting extent of germination vs. the logarithm        of analog T11-T22 concentration. Standard deviations are shown        in parentheses.    -   N/A, No activity under the conditions tested.

FIG. 10 is a graph of results from mice treated with CamSA orchenodeoxycholate. The graph shows no weight changes. Groups of fivemice were treated with DMSO (□), 50 mg/kg chenodeoxycholate (⋄), 50mg/kg CamSA (Δ), or 300 mg/kg CamSA (∘). Animal weight was obtaineddaily.

FIG. 11 displays graphic results of protection of mice from CDI bydifferent bile salts. The data is shown in a Kaplan-Meier survival plotfor C. difficile infected mice treated with DMSO (⋄), 300 mg/kgtaurocholate (Δ), 50 mg/kg chenodeoxycholate (∘), 50 mg/kg CamSA. (♦),or 300 mg/kg ethylcholate (X). Non-infected animals were used as control(□).

FIG. 12 shown graphic representations of signs of severity for C.difficile infected animals treated with different bile salts.Non-infected animals were used as control (panel A). Animals challengedwith C. difficile spores were treated with three doses of DMSO (panelB), taurocholate (panel C), chenodeoxycholate (panel D), CamSA (panelE), or ethylcholate (panel F). The severity of CDI signs was scoredusing the Rubicon scale discussed above.

FIG. 13 is a graph that shows that CamSA does not affect vegetativebacterial growth. E. coli DH5α (□), B. longum (∘), L. gasseri (Δ), andC. difficile (⋄) were incubated in media supplemented with 0 or 10 mMCamSA. The OD₅₈₀ was recorded at 0, 1, 2, 3, 4, 6, and 8 hours. Growthinhibition was determined by subtracting optical density ofCamSA-treated cultures from untreated control cultures.

FIG. 14 graphically shows that CamSA is not toxic to mammalian cells.Marine macrophages J774A.1 were treated with DMSO (panel A), 10% ethanol(panel B), or 200 μM CamSA (panel C). Cell viability was determined bytrypan blue dye exclusion staining.

FIG. 15 graphically shows distribution of C. difficile spores in the GItract of CamSA-treated animals. The stomach (St), duodenum (Du), jejunum(Je), and ileum (Il) showed negligible amounts of spores compared to thececum (Ce) and colon (Co).

FIG. 16 is graphic evidence that CamSA protects mice from CDI.Comparison of CDI sign severity after 48 hours (white bars) and 72 hours(black bars) of animals challenged with C. difficile spores and treatedwith DMSO, 300 mg/kg taurocholate (TC), 50 mg/kg chenodeoxycholate(CDCA), 50 mg/kg CamSA, or 300 mg/kg ethyl cholate (EC). Non-challenged(NC) animals were used as controls. Clinical endpoint was set as >6 inthe CDI sign severity scale (dashed line). None of the animals in theDMSO and EC groups survived to 72 hours post-challenged. Standarddeviations represent at least five independent measures.

TABLE 3 A→B^(b) B→A^(c) Efflux Compound (10⁻⁶ cm s⁻) (10⁻⁶ cm s⁻)Ratio^(d) Comment^(e) Ranitidine 0.3 1.8 5.3 Low permeability controlWarfarin 42.9 16.0 0.4 High permeability control CamSA 0.0 10.9 >2 LowPermeability. Efflux substrate ^(a)All tests were performed at 10 μMfinal concentrations and equilibrated for two hours ^(b)Apical tobasolateral apparent permeability (P_(app)) ^(c)Basolateral to apicalapparent permeability (P_(app)) ^(d)Efflux ratio (RE) >2 indicates asignificant efflux activity, an indication of potential substrate forPGP or other active transporters ^(e)Permeability ranking: Low (P_(app)< 0.5), Moderate (0.5 < P_(app) < 5), High (P_(app) > 5)

FIG. 17 is a time line model fir CDI onset in mice. C. difficile spores(black circles) that are ingested by the host. Spores rapidly transitthrough the upper GI tract and colonize the colon and cecum. Sporeshedding begins less than 2 hours post-ingestion. Between 6 and 9 hoursafter ingestion sufficient numbers of spores germinate to establishinfection. The outgrowing C. difficile cells (white circles) proliferatein the lower intestine, are shed, and can re-sporulate. A small amountof ingested spores remain in the lower intestine for more than 96 hourspost ingestion.

CamSA had no Observable Adverse Effects on Mice.

To determine the acute toxicity of CamSA to mice, we used the fixed doseprocedure. No physical adverse effects or weight loss were observed whenCamSA was administered for three consecutive days at doses up tosaturating 300 mg/kg. A 300 mg/kg dose of chenodeoxycholate causedimmediate death, probably due to observed precipitation ofchenodeoxycholate upon interaction with mouse saliva and gastric juice.Chenodeoxycholate at 50 mg/kg did not cause any observable side effects.

Prevention of CDI by Bile Salt Analogs.

As previously reported, when mice were challenged with 10⁸ CFU of C.difficile spores, severe CDI signs developed and all animals reachedclinical endpoint by 48 hours post-challenge. The large (10⁸ CFUs)inoculum of spores ensured synchronized CDI onset and fast CDI signprogression. Mice treated with up to 300 mg/kg taurocholate or ethylcholate also developed severe CDI and signs were undistinguishable fromuntreated animals. Mice treated with 50 mg/kg chenodeoxycholatedeveloped moderate to severe signs of CDI, but onset was delayed by 24hours. In contrast, all animals treated with 50 or 300 mg/kg CamSAshowed no sign of CDI and were undistinguishable from uninfectedanimals. All asymptomatic animals remained free of CDI signs for atleast 14 days post-challenge.

Stability of CamSA.

CamSA is a taurocholate analog with an amide bond linking cholic acid tometa-aminobenzene sulfonic acid. To be effective, CamSA must survive thechanging environments of the GI tract. To test for stability, CamSA wasincubated in artificial gastric juice and intestinal juice. Nodegradation of CamSA was evident even after 24 hours incubation underboth conditions (data not shown).

Bacterial bile salt hydrolases (BSHs) deconjugate primary and secondarybile salts. B. longum and L. gasseri are two intestinal bacteriacommonly used as test strains for BSH production. After incubation witha culture of B. longum for 24 hours, CamSA and taurocholate are bothhydrolyzed to cholic acid at similar rates. CamSA and taurocholate areless sensitive to degradation by BSHs secreted by L. gasseri. Less than30% of either CamSA or taurocholate was hydrolyzed to cholic acid after24 hours. E. coli does not produce BSH and both CamSA and taurocholatewere stable after 24 hour incubation with E. coli cultures (data notshown). CamSA was not degraded in growth medium alone.

CamSA Caco-2 Permeability.

To prevent C. difficile spores from germinating, CamSA needs to beretained in the intestinal lumen. Caco-2 monolayers serves as an invitro surrogate assay for intestinal permeability, absorption, andmetabolism. CamSA was studied in a Caco-2 permeability assay anddisplayed an apical to basolateral apparent permeability coefficient(P_(app)) of <10⁻⁶cm/s and basolateral to apical P_(app) of 10.9×10cm/s. The efflux ratio suggests that CamSA is a substrate for activetransport. In both assays, CamSA was recovered at 100% indicating lowbinding, accumulation, and metabolism by Caco-2 cells.

Effect of CamSA on Bacterial Growth.

E. coli, B. longum, and L. gasseri are indigenous mammalian gut bacteriaand are continuously exposed to bile salts. As expected, growth of thesebacteria was unaffected by the presence of CamSA in the growth medium.C. difficile cells also grew normally in the presence of CamSA.

Cytotoxicity of CamSA.

Cell viability was qualitatively determined by visual observation ofrounded/detached cells and trypan blue staining. CamSA-treated Vero,Caco-2, and macrophage cells appeared healthy and were undistinguishablefrom DMSO-treated cells. Cell viability was also quantitativelydetermined by ATP production. Vero and Caco-2 cells treated with 50 or200 μM CamSA produced ATP at similar levels to healthy control cells.

CamSA Protection of Vero and Caco-2 Cells:

Spent media from outgrowing C. difficile spores killed Vera cells in adose-dependent manner. These data are consistent with previous reportsindicating that vegetative C. difficile secretes cell-killing toxinsduring growth. When C. difficile spores were incubated in mediumcontaining 200 μM CamSA, bacterial growth was reduced but noteliminated. As expected, spent media from CamSA treated cultures wereless effective at killing epithelial cells. Similar results wereobserved for Caco-2 cell cultures (data not shown).

Timing of CDI Onset.

To determine the onset of CDI in mice, animals were challenged with C.difficile spores and treated with 300 mg/kg CamSA between 0 and 12 hourspost-challenge. All animals treated with CamSA up to 6 hourspost-challenge were fully protected from CDI. In contrast, all animalstreated with CamSA at 9 or 12 hours post-challenge developed severe CDIundistinguishable from untreated mice and reached the clinical endpoint48 hours post infection (FIGS. 5A and 5B).

Similar to previous reports, GI contents from animals with CDI signscontained almost exclusively C. difficile vegetative cells. Theseanimals started to excrete large amounts (>10×10⁵ CFUs) of vegetativecells reaching a maximum between 8 and 10 hours post spore challenge.Although some C. difficile spores were excreted in diseased animals, theamounts were negligible (<10% of vegetative CFUs) compared to the highamount of excreted vegetative cells.

FIG. 17 is a graphic representation of data showing that C. difficilespores accumulate in the cecum, colon, and feces of CamSA-treatedanimals. (A) Amount of C. difficile spores recovered at different timepoints following spore challenge from the cecum (white bars) and colon(black bars) of mice treated with 50 mg/kg CamSA. Students unpairedt-test was used to determine the significance of difference of means. *indicates recovered spores significantly below 72 hour levels (P=0.019;Students t-test). ** indicates recovered spores significantly below 72hour levels (P=0.049; Student's t-test). (B) Feces were collected fromcages housing five mice challenged with C. difficile spores and treatedwith 50 mg/kg CamSA. Closed bars represent C. difficile spores. Theamount of C. difficile vegetative cells in CamSA-treated animals wasnegligible (<10% compared to spore counts). Standard deviationsrepresent at least five independent measures. Recovered CFU andrecovered spores represent mean values from a pool of five animals.

Recovery of C. Difficile Cells and spores from Intestines and Feces ofCamSA-Treated Mice.

Similar to the hamster CDI model, ingested C. difficile spores narrowlylocalized to the cecum and colon of CamSA treated mice at every timepoint tested. A negligible amount of C. difficle was discovered in thesmall intestine and stomach. C. difficile spores remained in the cecumand colon for 72 hours after spore challenge (FIG. 17A). By 96 hours,the amount of spores recovered from the cecum and colon of CamSA treatedanimals decreased almost tenfold, from greater than 12×10⁵ to less than2×10⁵ CFUs.

Consistent with the results from intestinal content, the feces ofCamSA-treated animals contained almost exclusively spores (FIG. 17B). Inthese animals, excretion of ingested C. difficile spores started 2 hourspost-challenge and continued until at least 96 hours post-challenge. Infact, by 120 hours post-challenge, the sum of excreted C. difficilespores was quantitatively identical to the number of spores given bygavage.

FIG. 19 is a graphic representation of the stability of CamSA andtaurocholate towards bile salt hydrolases. CamSA (white bar) andtaurocholate (black bars) were incubated with cultures of B. longum or Lgasseri. Percent conjugated bile salts were derived by dividing theintensity of TLC spots obtained at different times by the intensity ofthe TLC spot obtained at the beginning of incubation (time 0). Time 0was set at 100% and is not shown for clarity. Standard deviationsrepresent at least five independent measures.

FIG. 20 is a graphic representation of the cytotoxicity of CamSA: Verocells (white bars) or Caco-2 cells (black bars) were incubated overnightwith 10% DMSO, 10% EtOH, 50 μM CamSA or 200 μM CamSA. Cell viability wasdetermined with the CellTiter Glo viability kit. The luminescence signalfrom DMSO-treated cells was undistinguishable from untreated cells andwas set as 100% cell viability. Percent survival for other conditionswas calculated relative to untreated cells. Error bars representstandard deviations from at least five independent measurements.

FIG. 21 is a graphic representation of inhibition of C. difficile toxinproduction by CamSA treatment. C. difficile spores were incubatedovernight in media containing 0 μM CamSA (white bars) or 200 μM CamSA(black bars). The resulting spent media were added to Vero cell culturesand incubated for 24 hours. Cell viability was determined with theCellTiter Glo viability kit. The luminescence signal from untreatedcells was set as 100 cell viability. Percent survival for otherconditions was calculated relative to untreated cells. Error barsrepresent standard deviations from at least five independentmeasurements.

FIG. 22 is a graphic representation that CDI is established between 6and 9 hours post-infection. (A) Survival of infected mice at 48 hoursafter challenge with C. difficile spores. Mice were treated with 300mg/kg CamSA at 0, 6, 9, or 12 hours post-challenge. (B) Comparison ofCDT severity after 24 hours (white bars) and 48 hours (black bars) foranimals challenged with C. difficile spores and treated with 300 mg/kgCamSA at 0, 6, 9, or 12 hours post-challenge. Clinical endpoint was setas >6 in the CDI sign severity scale (dashed line). (C) C. difficilevegetative cell, count in feces of untreated, diseased animals. Feceswere collected from cages housing five untreated mice challenged with C.difficile spores. Open bars represent C. difficile vegetative cells. Theamount of C. difficile spores excreted by untreated animals wasnegligible (<10% of vegetative cell counts). Standard deviationsrepresent at least five independent measures. Recovered CFU andrecovered spores represent mean values from pools of five. animals.

1. A method of treating a patient to reduce risk of developingClostridium difficile-associated disease or reducing Clostridiumdifficile-associated disease in a mammalian subject comprisingadministering to a mammalian subject an effective amount of a compoundhaving a structure represented by a formula:

wherein each of R¹ and R² is independently selected from hydrogen,halogen, —OH, —CN, —NH₂, —CO₂H, C1-C6 alkylamino, (C1-C6) (C1-C6)dialkylamino and C1-C6 alkyl optionally substituted with a groupselected from halogen, —OH, and —CN; wherein R³ is selected fromhydrogen halogen —CN CO₂H and C1-C6 alkyl optionally substituted with agroup selected from halogen, —OH, and —CN; and wherein R⁴ is selectedfrom O and S and R⁵ is selected from —O(CH₂)₂SO₃H, —NHCH₂SO₃H,—NH(CH₂)₃SO₃H, —NH(CH₂)₂CO₂H, —NH(CH₂)₃CO₂H, —NH(o-Ph)SO₃H,—NH(m-Ph)SO₃H, —NH(p-Ph)SO₃H, —NH(CH₂)₂SO₂H, —NH(CH₂)₂CONH(CH₂)₂CO₂H,and —NH(CH₂)₃CONH(CH₂)₃CO₂H; or wherein R⁴ is S and R⁵ is selected from—NH(CH₂)₂SO₃H and —NHCH₂CO₂H, provided that when each of R¹ and R² is—OH, R³ is hydrogen, and R⁴ is O, then R⁵ cannot be —NH(m-Ph)SO₃H, or aderivative thereof. 2-4. (canceled)
 5. A compound having a structurerepresented by a formula:

wherein each of R¹ and R² is independently selected from hydrogen,halogen, —OH, —CN, —NH₂, —CO₂H, C1-C6 alkylamino (C1-C6) (C1-C6)dialkylamino and C1-C6 alkyl optionally substituted with a groupselected from halogen, —OH, and —CN; wherein R³ is selected fromhydrogen halogen, —CN, —CO₂H, and C1-C6 alkyl optionally substitutedwith a group selected from halogen, —OH, and —CN; wherein R⁴ is selectedfrom O and S and R⁵ is selected from —NH(o-Ph)SO₃H, —NH(m-Ph)SO₃H,—NH(CH₂)₂SO₂H, —NH(CH₂)₂CONH(CH₂)₂CO₂H, and —NH(CH₂)₃CONH(CH₂)₃CO₂H; orwherein R⁴ is S and R⁵ is selected from —NHCH₂SO₃H, —NH(CH₂)₂SO₃H,—NH(CH₂)₃SO₃H, —NH(p-Ph)SO₃H, —NHCH₂CO₂H, —NH(CH₂)₂CO₂H, —NH(CH₂)₃CO₂H,and —O(CH₂)₂SO₃H, provided that when each of R¹ and R² is —OH, R³ ishydrogen, and R⁴ is O, then R⁵ cannot be —NH(m-Ph)SO₃H, or a derivativethereof. 6-8. (canceled)
 9. The method of claim 1, wherein each of R¹and R² is —OH.
 10. The method of claim 1, wherein R³ is hydrogen. 11.The method of claim 1, wherein R⁴ is O.
 12. The method of claim 1,wherein R⁵ is selected from —NH(o-Ph)SO₃H, —NH(m-Ph)SO₃H, —NH(p-Ph)SO₃H,—NH(CH₂)₂SO₂H, —NH(CH₂)₂CONH(CH₂)₂CO₂H, and —NH(CH₂)₃CONH(CH₂)₃CO₂H. 13.The method of claim 1, wherein R⁵ is selected from —NH(o-Ph)SO₃H,NH(p-Ph)SO₃H, —NH(CH₂)₂SO₂H, —NH(CH₂)₂CONH(CH₂)₂CO₂H, and—NH(CH₂)₃CONH(CH₂)₃CO₂H.
 14. The method of claim 1, wherein R⁵ isselected from —NH(CH₂)₂SO₂H, —NH(CH₂)₂CONH(CH₂)₂CO₂H, and—NH(CH₂)₃CONH(CH₂)₃CO₂H.
 15. The method of claim 1, wherein the compoundhas a structure represented by a formula selected from:


16. The method of claim 1, wherein the compound has a structurerepresented by a formula:


17. The method of claim 1, wherein the compound has a structure selectedfrom:


18. The compound of claim 5, wherein each of R¹ and R² is —OH.
 19. Thecompound of claim 5, wherein R³ is hydrogen.
 20. The compound of claim5, wherein R⁴ is O.
 21. The compound of claim 5, wherein R⁵ is selectedfrom —NH(o-Ph)SO₃H, —NH(m-Ph)SO₃H, —NH(CH₂)₂SO₂H,—NH(CH₂)₂CONH(CH₂)₂CO₂H, and —NH(CH₂)₃CONH(CH₂)₃CO₂H.
 22. The compoundof claim 5, wherein R⁵ is selected from —NH(CH₂)₂SO₂H,—NH(CH₂)₂CONH(CH₂)₂CO₂H, and —NH(CH₂)₃CONH(CH₂)₃CO₂H.
 23. The compoundof claim 5, wherein the compound has a structure represented by aformula selected from:


24. The compound of claim 5, wherein the compound has a structurerepresented by a formula:


25. The compound of claim 5, wherein the compound has a structurerepresented by a formula:


26. The method of claim 1, wherein the compound has a structure selectedfrom: